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Analysis of free galactose contents during cold storage of four apple cultivars, in thermally treated… Jim, Vickie Jin Wai 2002

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ANALYSIS O F F R E E G A L A C T O S E CONTENTS DURING C O L D S T O R A G E O F F O U R A P P L E C U L T I V A R S , IN T H E R M A L L Y T R E A T E D A P P L E S A N D G R E E N B E A N S , A N D IN C L E A R A P P L E J U I C E S P R O D U C E D USING D I F F E R E N T E N Z Y M A T I C AIDS By  V I C K I E JIN W A I J I M B.Sc. (Diet.) University of British Columbia, Vancouver, 1999.  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Food Science We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A September 2002 © V i c k i e Jin Wai Jim, 2002.  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date  Abstract Dietary control of galactose is the only recourse available to patients with galactosemia because there is no cure or drug that can control the disease. However, it is impossible to eliminate all sources of galactose because it is widely available in foods. Fruits and vegetables, which were previously considered to be safe for consumption, have been shown to contain small but significant amount of galactose that can cause complications. Currently, there is limited information on how extended storage and different processing techniques can affect the free galactose content in fruits and vegetables. Therefore, this research investigated how free galactose is affected by cultivar difference and storage time in 4 varieties of apples; thermal processing in apples and green beans; and enzymatic preparations used in clear apple juice production. Sugars were extracted from produce with 80% ethanol and free galactose contents was determined by gas chromatography where galactose and other sugars were derivatized into oxime-trimethylsilyl derivatives for monosaccharides and trimethylsilyl derivatives for sucrose before analysis. Different cultivars of apples were shown to have different free galactose contents and to exhibit different characteristics in changes in amount of free galactose during the 9-month storage. Red Delicious and Braeburn apples did not show significant increase in free galactose concentration over the course of the 9-month storage period. Spartan and Fuji showed increase after 3 and 6 months of storage respectively and the increases persisted until the end of the storage study. Blanching caused leaching of free galactose into the processing water and canning to commercial sterility partially solubilized pectin polymers and release galactose from  the pectin side chains but the liberated galactose was also solubilized into processing water, therefore, free galactose concentration in the plant tissue decreased. Canning to double commercial sterility increased the free galactose concentration in plant tissue. This may have been due to the release of galactose from hemicellulosic polysaccharides at this increased thermal treatment, and its entrapment inside the cellulosic matrix of the plant cell wall. The use of a liquefaction enzymatic preparation in clear apple juice production caused the free galactose content in the apple juice increased by 14.62mg /lOOmL compared to the control juice because cell wall polymers were completely broken down leading to the release of free galactose into the juice. Clarification aids, in contrast, only caused a slight increase in free galactose concentrations due to its selective action on soluble pectin. Information gained from this project will allow dietitians to better manage the diet and cooking practices of galactosemic patients to reduce intake of galactose and alleviate the occurrence of complications.  iii  TABLE OF CONTENTS  Page  ABSTRACT  ii  T A B L E OF CONTENTS  tV  LIST OF T A B L E S . . . . ,  ix  LIST OF FIGURES  X  ACKNOWLEDGEMENTS  XU  1. INTRODUCTION  1  2. PURPOSE OF R E S E A R C H 2.1. Research objectives 2.2. Hypotheses  4 4 4  3. L I T E R A T U R E R E V I E W 3.1. Galactose 3.1.1. Sources of galactose in the diet 3.1.2. Digestion, absorption and metabolism of galactose  6 6 6 7  3.2. Galactosemia 3.2.1. Galactosemia 3.2.2. Dietary management of galactosemia 3.2.3. Limitations of current treatment  10 10 11 13  3.3. Fruit ripening 3.3.1. Plant cell wall polysaccharides 3.3.2. Structural changes of plant cell walls during ripening 3.3.3. Enzymes involved in fruit ripening  13 14 15 16  3.4. Long-term storage of fruits and vegetables 3.4.1. Cold storage in regular atmosphere  17 19  3.5. Heat processing 3.5.1. Blanching 3.5.2. Sterilization  19 19 20  3.5.2.1. pH classification of canned foods 3.5.2.2. D and z values 3.5.2.3. Determining lethality of thermal processes 3.5.3. Effects of heat treatment on cell wall polysaccharides 3.6. Enzymes used in fruit juice production 3.6.1. Fruit juice clarification 3.6.2. Enzyme treatment of pulp to increase juice yield (mash treatment and liquefaction) 3.6.2.1. Increasing press capacity: mash treatment 3.6.2.2. Liquefaction 3.7. Apples  20 21 22 24 25 26 26 27 28  Apple production Composition Storage Processing 3.7.4.1. Canning 3.7.4.2. Juice production  29 30 30 30 31 31 32  3.8. Carbohydrate analysis 3.8.1. Extraction of sugars 3.8.2. Quantification of sugar mixtures 3.8.3. Chromatography 3.8.4. Gas chromatography 3.8.4.1. Principles 3.8.4.2. Sample preparation—derivatization 3.8.4.3. Columns 3.8.4.4. Detectors  32 32 33 34 34 35 36 38 39  3.7.1. 3.7.2. 3.7.3. 3.7.4.  4. M A T E R I A L S A N D METHODS  41  4.1. Storage trial 4.1.1. Produce selection 4.1.2. Storage condition 4.1.3. Sample selection  41 41 41 41  4.2. Thermal processing 4.2.1. Blanching 4.2.2. Canning 4.2.2.1. Canning of apples 4.2.2.2. Canning of green beans 4.2.2.3. Heat penetration determination of cold spot during canning  42 42 43 43 43 44  V  4.3. Juice production 4.3.1. Juice 1: Control juice (no enzyme preparation added) 4.3.2. Juice 2: Juice with liquefaction enzymes added 4.3.3. Juice 3: Juice with liquefying enzymes and clarification enzymes added 4.3.4. Juice 4: Juice with clarification enzymes added 4.3.5. Brix and pH determination 4.4. Sugar extraction  44 45 45 45 46 46 46  4.5. Sugar derivatization and quantification 48 4.5.1. Alditol acetate derivatization 48 4.5.1.1. Derivatization procedures 48 4.5.1.2. Gas chromatography for alditol acetate derivatives 49 4.5.2. Trimethylsilyl (TMS) derivatization 49 4.5.2.1. Derivatization procedures 49 4.5.2.2. Gas chromatography for TMS-oxime and TMS derivatives 50 4.6. Statistical analysis  5. RESULTS A N D DISCUSSION 5.1. Derivatization methods 5.1.1. Alditol acetate derivatization 5.1.2. Trimethylsilylated-oxime (TMS-oxime) derivatives 5.1.3. Problems in sampling method  51  53 53 53 59 68  5.2. Storage study of different varieties of apples 73 5.2.1. Conditions of apples during storage 73 5.2.2. Free galactose in apples 73 5.2.3. Free arabinose in apples 78 5.2.4. Sucrose in apples 82 5.2.5. Effects of varietal differences and storage on the amounts of free galactose, arabinose and sucrose in apples 86 5.3. Thermal treatments of apples and green beans 88 5.3.1. Lethality calculation 88 5.3.2. Free galactose, arabinose and sucrose concentrations in Fuji apples after different kinds of thermal processing 90  vi  5.3.3. Free galactose, arabinose and sucrose concentrations in green beans after different kinds of thermal processing 90 5.3.4. Effects of thermal processing on the free galactose, arabinose and sucrose contents in apples and green beans...93 5.4. Enzymatic aids used in juice production 97 5.4.1. Juice characteristics 97 5.4.2. Free sugars contents in clear, amber coloured apple juice produced with the addition of different enzymatic preparations 99 5.4.3. Effects of addition of different enzyme preparations to the free sugars concentrations in apple juice 102 5.4.4. Potential effect of physiological age of apples on juicing and canning 104 5.5. Implications of this work for dietary management for galactosemic patients  106  6. CONCLUSIONS  Ill  REFERENCES  113  APPENDIX A  123  I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI APPENDIX B 1  124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141  V i i  II III IV V VI VII VIII IX X XI APPENDIX C I II III IV  142 143 144 145 146 147 148 149 150 151 152 153 154 155 156  Viii  LIST OF TABLES Table 1. The % recovery of different amounts of galactose standard added to stored Fuji apples (11 months at 4°C) 70 Table 2. Free galactose content of Fuji apples that had been stored for 11 months in regular atmosphere at 4°C  72  Table 3. Free galactose content (mg/lOOgfw) of 4 different apple cultivars stored for 0, 3, 6, 9 months in regular atmosphere at 4°C 74 Table 4. 2-way analysis of variance of free galactose content in 4 apple cultivars stored in regular atmosphere at 4°C for 9 months. Samples from each cultivar were taken after 0, 3, 6, 9 months of storage 76 Table 5. Free arabinose content expressed as peak area ratio (peak area of arabinose/ peak area of IS) of 4 different varieties of apples stored at 4°C in regular atmosphere for 0, 3, 6, 9 months 79 Table 6. 2-way analysis of variance of free arabinose content (as peak area ratio) in 4 apple cultivars stored in regular atmosphere at 4°C for 9 months. Samples from each cultivar were taken after 0, 3, 6, 9 months of storage 81 Table 7. Peak area ratio of sucrose (sucrose peak area/ internal standard peak area) as a TMS derivative of 4 different varieties of apples stored at 4°C in regular atmosphere 83 Table 8. 2-way analysis of variance of free sucrose content (as peak area ratio) in 4 apple cultivars stored in regular atmosphere at 4°C for 9 months. Samples from each cultivar were taken after 0, 3, 6, 9 months of storage 85 Table 9. Amount of lethal heat treatment canned apple slices and canned green bean received calculated with Improved General Method 89 Table 10. Free galactose, arabinose contents and sucrose peak area ratio in fresh, blanched and canned Fuji apples  91  Table 11. Free galactose, arabinose contents and sucrose peak area ratio in fresh, blanched and canned green beans  92  Table 12. Total soluble solids (°Brix), p H and yield of Red delicious juice produced by the addition of different enzymatic preparation aids 98 Table 13. Free galactose, arabinose contents and sucrose peak area ratio in clear apple juice produced with the addition of different enzymatic preparations 101  LIST OF FIGURES Figure 1. Glycogensis, by which the formation of glycogen from glucose occurs. Galactose can be converted to glucose via a number of reactions catalyzed by galactokinase, galactose-l-phosphate uridyltransferase and UDP-galactose epimerase 9 Figure 2. Separation of alditol acetates of a standard mixture of allose (internal standard) and galactose, each with a concentration of 0.1 mg/ mL. Peak identification: 1, allose; peak area=50519; 2, galactose; peak area=50389 54 Figure 3. Galactose standard curve (0.01 mg/ mL to 4.0 mg/ mL) produced by comparing the peak area ratio of galactose: allose (y-axis) and the weight ratio of galactose: allose standard (x-axis). Response factor of galactose compared to allose is 0.8914 with r =0.9952 55 2  Figure 4. Gas chromatogram of monosaccharides extracted from Spartan apples derivatized into their alditol acetate derivatives. Peak identification: 1, allose; 2, galactose 57 Figure 5. Gas chromatogram of monosaccharides extracted from Spartan apples spiked with 2mg/mL galactose standard, derivatized into their alditol acetate derivatives 58 Figure 6. Separation of TMS-oximes and TMS derivatives of a mixture of neutral sugars by gas chromatography. Peak identification: 1, fructose (lmg/mL); 2, galactose (O.OOlmg/mL); 3, glucose (lmg/mL); 4, phenyl-P-glucopyranoside (internal standard) (O.lmg/mL) 60 Figure 7. Enlarged view of gas chromatogram of fructose, galactose and glucose standard mixture as TMS-oximes derivatives. Peak identification as stated in Figure 6 61 Figure 8. Gas chromatogram of 0.05mg/mL galactose and O.lmg/mL phenyl-Pglucopyranoside (internal standard). Peak identification: la, galactose major peak; lb, galactose minor peak; 2, phenyl-P-glucopyranoside 63 Figure 9. Gas chromatogram of 0.2mg/mL arabinose and O.lmg/mL phenyl-pglucopyranoside (internal standard). Peak identification: la, arabinose major peak; lb, arabinose minor peak; 2, phenyl-P-glucopyranoside 64 Figure 10. Galactose standard curve produced by comparing the weight ratio of galactose (mg) to internal standard (IS) (mg) (x-axis) and the peak area of galactose to IS (y-axis) as TMS-oximes derivatives. Galactose has a response factor of 1.2307 with reference to IS. The standard curve has a r =0.9906 65 2  X  Figure 11. Gas chromatogram of sugars extracted from Fuji apples stored for 9 months at 4°C. Sugars were derivatized into TMS-oxime and TMS derivatives. Peak identification: 1, arabinose; 2, fructose; 3, galactose; 4, glucose; 5, phenyl-pD-glucopyranoside (internal standard); 6, sucrose. Peak area of galactose= 675 and peak area of phenyl-|3-D-glucopyranoside=12054 66 Figure 12. Enlarged image of sugars extracted from Fuji apples (Figure 11) as T M S oximes and TMS derivatives, showing the shape and area of galactose peak (#3). Peak identification as stated in Figure 11 67 Figure 13. Gas chromatograms (enlarged—showing from 10 minutes to 20 minutes) of sugars extracted from Fuji apples stored for 11 months at 4°C spiked with 0 (Figure 13a), 0.577mg (Figure 13b), 1.009mg (Figure 13c) and 2.184mg (Figure 13d) of galactose standard. Galactose was added before extraction of sugars with 80% ethanol. Peak identification as stated in Figure 11 69 Figure 14. Effect of storage in regular atmosphere at 4°C on the free galactose content (mg/lOOgfw) in 4 varieties of apples. Data points are shown as mean ± standard deviation 77 Figure 15. Effect of storage in regular atmosphere at 4°C on the free arabinose content (expressed as arabinose peak area/ internal standard (IS) peak area) in 4 varieties of apples. Data points are shown as mean ± standard deviation 80 Figure 16. Effect of storage in regular atmosphere at 4°C on the free sucrose content (expressed as sucrose peak area/ internal standard (IS) peak area) in 4 varieties of apples. Data points are shown as mean ± standard deviation 84  Xi  ACKNOWLEDGMENTS  I would like to thank Dr. Christine Seaman for her constant support, guidance and patience throughout this research. It is an honour to be able to work with you. I have learnt a lot from you both academically and intellectually. Thank you for being an inspiration. I would also like to thank my committee members: Dr. Tim Durance, Ms. Carol Hartnett and Dr. John Vanderstoep for sharing their knowledge, giving advises and devoting their time during this study. This research would not be possible without the help of Mr. Sherman Yee. Thanks not only for sharing your expertise in Food Science but also for your continuous encouragements and care. Thanks to Mrs. Valerie Skura and Ms. Brenda Barker for fulfilling all my requests and solving all my emergency problems. I'm also very grateful to have wonderful classmates and friends from this department who share laughters with me and keep me company during long nights and weekends in the lab. Finally, thanks to my family and best friends for their never ending support and believing in me even when I do not believe in myself. Thanks for giving me the freedom to choose my own path.  Ki)  1. Introduction Galactosemia is an inborn error of metabolism that results from defects in any of several enzymes that are involved in the metabolism of the sugar, galactose. Galactose1-uridyltransferase (GALT) is the most common defective enzyme that causes accumulation of galactose and galactose-1-phosphate and production of galactitol (Gropper et al, 1993). Galactosemia leads to detrimental complications including liver disease, central nervous system disturbances, developmental delay, learning disabilities, and in females, ovarian failure. There is virtually no information on a cure or drug therapy for the disease and dietary control of galactose remains the only recourse available to patients. The treatment sounds simple; however, the total elimination of galactose in the diet is unachievable due to the vast availability of galactose in nature. Previously patients were advised to include large amounts of fruits, vegetables and lentils in their diet. The safety of this recommendation has been challenged after studies by Gross and Acosta (1991) and Gropper et al. (2000) showed that fruits and vegetables contain small but significant amounts of free galactose. It is estimated that patients on a traditional galactose restricted diet can consume greater than 500 mg of galactose from fruits and vegetables each day (Berry et al, 1993; Gross and Acosta, 1991). There have only been a few studies that have investigated the free galactose concentrations in foods. Gross and Acosta (1991) and Gropper et al. (2000) evaluated the free galactose contents of fresh fruits and vegetables; Gropper et al. (1993) and Gross et al (1995) examined baby food fruits and vegetables. They concluded that fruits and vegetables contain amounts of free galactose that may pose problems if these produce are  1  consumed daily. Although a number of fresh fruits and vegetables have been analyzed, free galactose content in different varieties of a single product have not been included in previous studies. Cultivar difference might affect the amount of free galactose in produce since it is known that different cultivars of a same product such as apples, have different amounts and composition of major sugars such as fructose and glucose (Fuleki et al, 1994), however, others, like onion, do not show cultivar differences (Ng et al, 1998). Galactose is mainly found bound to the side chains of cell wall polysaccharides like hemicellulose and pectin. During storage, these polysaccharides are solubilized and degraded, causing the bound galactose to be released from the side chains. It is well documented that during storage, galactose is lost from pectin polymers of the primary cell wall as the stored produce ripens (Redgwell et al, 1997; Kitagawa et al, 1995; Gheyas et al, 1998). However, it is not known if the galactose that is lost from pectic polysaccharides remains in the plant tissue as free galactose or is further metabolized. Thermal processing can also release galactose from cell wall polymers (Nyman et al, 1994). Pectins are solubilized and degraded as heat is applied and it is found that canning has the greatest influence. Cell wall polymers in carrots (Plat et al, 1988), green beans (Stolle-Smits et al, 1995) and apricots (Chitarra et al, 1989) were reported to be solubilized and degraded during canning and loss of galactose from these polysaccharides was also documented. Reported free galactose values for juices are not consistent. Apple juice has been reported to contain approximately 2 mg/ 100 mL (Gross et al, 1995) and up to 30 mg/ 100 mL (Fuleki et al, 1994). Such variation may be due to the addition of different types of cell wall degrading enzymatic preparations to help in pressing and clarification of  2  apple juice. These enzymatic aids increase juice yield and/ or clarify the juice by breaking down the cell wall polysaccharides to reduce its water holding capacities. In doing so, galactose is released as pectins and hemicellulose are degraded. This study was intended to gain information on how varietial differences, storage and processing (thermal and juice production) affect free galactose concentrations in foods thus allowing dietitians to give better recommendations to patients as to selecting the appropriate foods. Apple is mainly used in this study as it fulfills all the mentioned factors that can affect free galactose concentrations mentioned above. There are many different cultivars, that can be easily identified, available in the market place; they are also stored for later consumption due to its one time harvest in the fall each year. Furthermore, apple is one of the major fruit that is canned and produced into clear juices.  3  2. Purpose of research  2.1. Research objectives This research project was developed to understand how varieties, storage and processing (thermal treatments and enzymatic aids used in clear juice production) can affect the amount of free galactose in foods. This information hopefully can assist patients with galactosemia to make better food choices to reduce the likelihood of complications. More specifically, this research aims: 1) To determine the soluble galactose content of apples kept under cold storage (4°C) and to establish the influence of extended storage and the physiological aging on free galactose levels. 2) To evaluate the effects of different severity of heat treatments (e.g. blanching and canning) on the free galactose content of high p H (green beans) and low p H (apples) produce. 3) To analyze the effect of different enzymatic preparations used for clarification and liquefaction during juice manufacture on free galactose content in clear apple juice. 4) To compare the free galactose content of different apple cultivars.  2.2. Hypotheses 1) H : There is no difference in the free galactose content in different varieties of apples. 0  H : Different varieties of apples have different free galactose content. a  2) H : Free galactose concentration in apples is independent of the length of storage 0  time at 4°C.  4  H : Free galactose concentrations in apples is influenced by the length of time stored a  in cold storage. 3) H : Free galactose levels in produce are not influenced by different heat treatments 0  applied. Ff : Different heat treatments have different impact on the free galactose levels in a  produce. 4) H : Different enzymatic preparations added to aid in the production of clear apple 0  juice have no effect on the amount of free galactose in the clear juice produced. Ff : Amounts of free galactose in clear apple juice produced by different processing a  techniques are different due to different enzymatic preparations added.  5  3. Literature Review  3.1. Galactose 3.1.1. Sources of galactose in the diet Galactose is a hexose that only exists in a D configuration in nature. It has a carbonyl group at the end of the carbon chain (C6) making it an aldehyde, therefore, galactose is classified as an aldose. Galactose can be found in 3 forms in nature—bound to glucose as lactose, free form, and bound to other polysaccharides, proteins and lipids. Galactose is found principally (3-1,4 linked to glucose as part of the digestible disaccharide lactose. Milk and milk products are the only natural sources of lactose in the diet. This includes yogurt, whey, casein, whey solids, curds, and texturized proteins. Fermented dairy products and aged cheese have been shown also to contain lactose (Harvey et al, 1981), although it is a common misconception that all lactose has been converted to lactic acid. In addition, lactose is added to a lot of products such as baked goods, dry mixes, confections and batter mixes to improve flavour, texture, body, viscosity and mouthfeel. Lactose can also be found as an extender in many over-thecounter and prescription drugs (Acosta and Gross, 1995; Elsas and Acosta, 1994). Galactose can also exist in minor amounts in free form (i.e. not bound to other sugar or compound) in many plant tissues, including grains, fruits, and vegetables (Gross and Acosta, 1991; Gropper et al, 2000). Amounts range from <0.5 mg/100 g fresh weight in radish, spinach and mushroom to 26 mg/ 100 g fresh weight in blueberries, honeydew melon and 35.4 mg/ 100 g fresh weight in persimmon.  6  The primary cell wall of plants and their various edible parts consist of many polysaccharides and some of them contain galactose. These galactose containing plant cell wall polysaccharides include galactose-containing oligosaccharides, galactolipids, pectin, galactan and hemicellulose. Essentially all plant tissues contain galactose in various forms, including a-galactosides (e.g. raffinose, stachyose, and verbascose), galactolipids and various other glycosidic linkages in arabinogalactan proteins and cell wall polysaccharides (Acosta and Gross, 1995). Galactan contains a repeating chain of Dgalactose in 3-1,4 linkage and it is primarily found as part of the pectic polymer fraction and hemicellulose as side chains (Brinson and Dey, 1985). Other than bound to other carbohydrates, galactose can also be found bound to proteins and lipids. It is a component of glycoproteins found in plants and edible meats, such as brain, kidney and liver and is found in galactosylcerebrosides and gangliosides. Glycolipids containing galactose are also found in many foods. The galactose can be attached to glycerol in mono- and digalactosyldiacylglycerol via {3-linkage. The second galactose residue in digalactosyldiacylglycerol is in an cc-linkage. 3.1.2. Digestion, absorption and metabolism of galactose Although various forms of galactose are widely present in foods, they are not all digestible by human and available for absorption. Hydrolysis of the a-galactosides by digestive enzymes within the human gastrointestinal tract is doubtful (Elsas and Acosta, 1994). The P-galactosidase (EC 3.2.1.23) present in the human small intestine is mainly for hydrolysis of lactose into equal amounts of galactose and glucose. Although hydrolysis of other types of P-1,4 linked galactans by p-galactosidase (EC 3.2.1.23) is possible, P-1,4 galactose-containing oligosaccharides are very rare in foods (Dey, 1985);  7  therefore their digestion is not usually considered a significant factor in galactose consumption in humans. There are also bound galactose residues in pectins and hemicellulose; however, whether humans can digest and metabolize these (3-1,4 galactose derivatives and galactose-containing side chains attached to pectin and hemicellulose is still controversial. Galactose in the small intestine is absorbed into the mucosal cells by active transport where energy and a specific receptor are required. However, the exact nature of the glucose-galactose carrier is unclear (Groff et al, 1995). Following transport across the gut wall, the monosaccharides enter the portal circulation, wherein they are carried directly to the liver. Since glucose is the exclusive monosaccharide that can be used by the body for metabolism and the building block for glycogen, in order for galactose to be useful, it needs to be converted to glucose derivatives. Galactose is first phosphorylated. The transfer of the phosphate from ATP is catalyzed by galactokinase, and the resulting phosphate ester is at carbon 1 of the sugar. Galactose-1-phosphate can be converted to glucose-1-phosphate through the intermediates, uridine disphosphate (UDP)-galactose and UDP-glucose. The enzyme galactose-1-phosphate uridyltransferase transfers an uridyl phosphate residue from UDP-glucose to the galactose-1-phosphate, yielding glucose-1-phosphate and UDP-galactose. In a subsequent reaction catalyzed by epimerase, UDP-galactose can then be converted to UDP-glucose in which form it can be converted glucose-1-phosphate by the uridyl transferase reaction mentioned above, or it can be incorporated into glycogen by glycogen synthase (Figure 1). It can also enter the glycolytic pathway as glucose-6-phosphate, which can be hydrolyzed to free glucose in liver cells (Groff et al, 1995).  8  HOCH^  A  HO  0.  D-Galactose  a-D-Glucose-6-phosphate  ATP Phosphoglucomutase  Galactokinase  ADP  HOC^ HO A .OH  HOCHj  OH  OH Galactose-1 -phosphate  Glucose-1 -phosphate  UTP UDP-Glucosepyrophosphorylase PP=  O,  UDP-Glc:Gal-1-P uridyltransferase  < —  HOCH^ HO  HOCH^ H O < £ _ + ^ 0 - UDP  OH  UDP-Galactose Epirnerase  OH  UDP  Uridine diphosphate galactose  Uridine diphosphate glucose Glycogen Synthetase  HOCHj  HOCH,  Glycogen Chain  HO OH  OH  Figure 1. Glycogenesis, by which the formation of glycogen from glucose occurs. Galactose can be converted to glucose via a number of reactions catalyzed by galactokinase, galactose-l-phosphate uridyltransferase and UDP-galactose epirnerase (Source: Miller, 1998). 9  3.2. Galactosemia 3.2.1. Galactosemia Galactosemia is an autosomal recessive disorder, occurring in the United States with a variable frequency of between 1 in 18,000 and 1 in 70,000 (Elsas and Acosta, 1994). It may result from the defective function of the enzymes involved in converting galactose to glucose derivatives, namely: galactokinase, galactose-1-phosphate uridyltransferase, or uridine diphosphate galactose-4-epimerase (Gropper et al, 1993). Manifestations of galactosemia vary depending on the defective enzyme. Galactokinase deficiency results primarily in cataracts due to galactitol accumulation. In contrast, galactose-l-uridyltransferase is the most common defective enzyme, and results in accumulation of galactose and galactose-1-phosphate and production of galactitol (Berry et al, 1993). The gene that codes for galactose-1-phosphate uridyltransferase is located on chromosome 9. Many different mutations within this gene have been identified, and the most common mutation is called "Q188R" (Elsas II et al, 1995). If galactose-luridyltransferase deficiency is left untreated, patients develop severe haptic, renal and gastrointestinal manifestations which, can lead to death (Donnell et al, 1980). Despite early diagnosis and dietary therapy for galactose-1-phosphate uridyltransferase deficiency, chronic complications such as cataracts, gynecologic failure, speech and language delays, neurologic deficits, and failure to thrive in infants have been reported. In the past, only dietary lactose restriction was thought to be sufficient to reverse the early manifestations or to eliminate their expression in prospectively treated infants. However, with time, the chronic complications mentioned above emerge even in patients that follow strict diets. There are several theories to explain the poor outcome of this  10  disease. One of them is chronic intoxication with galactose either from endogenous source or consumed exogenously. However, whether the amount of sugar causing toxicity is produced endogenously by the breakdown of UDP-galactose or obtained exogenously from the diet has not yet been established. Endogenous self-intoxication could stem from the continuous formation of galactose-1-phosphate by cleavage of UDPgalactose, which is normally formed from glucose via UDP-glucose. Others thought that the intoxication might be associated with exogenous sources of galactose, due to ingestion of galactose from hidden sources such as free and/ or macromolecular-bound galactose in fruits and vegetables (Berry et al, 1995; Segal, 1995). Although the cause and optimal treatment to prevent these long-term poor outcomes has not been established, restriction of dietary galactose intake is imperative to prevent acute problems, such as liver and kidney damage, and to diminish risk of sepsis, and perhaps to minimize other long-term poor outcomes (Gropper et al, 2000). However, since galactose is widely found in foods, prevention of exposure to all exogenous galactose sources is impossible. 3.2.2. Dietary management of galactosemia Objectives of dietary management in galactosemia are to ameliorate or to prevent symptoms while providing adequate energy and nutrients for normal growth and development (Elsas and Acosta, 1994). Treatment of galactosemia, best instituted during the first week of life, involves removal of all sources of lactose and free galactose from the diet (Gross et al, 1995). Thus, infants with galactosemia typically receive a low galactose infant formula (e.g. formulas containing soy protein isolates) to initially meet nutrient needs (Elsas and Acostam 1994). Milk and dairy products are traditionally  11  eliminated from the diet because of their natural lactose content. At 4 to 6 months of age, baby cereals are added to the diet. Gross et al. (1995) reported boxed baby cereals containing only cereal were low in free galactose. However when baby fruit juices, baby food fruits and vegetables and solid foods are introduced to the diet, it is possible for problems to arise. Solid foods, when added to the diet, are widely restricted because of the expansive list of foods to which lactose has been added (Gropper et al., 1993). Additional restricted intake is recommended for sources of galactose include beans, legumes and organ meats such as brain, liver and kidney (Clothier and Davidson, 1983). It is certain that no individual with galactosemia can achieve a completely galactose-free diet due to its vast availability in nature. Patients were advised to include large amounts of lentils, fruits and vegetables to increase variety in the diet. The safety of this recommendation has been challenged by Gross and Acosta (1991) and Gropper et al. (2000) who showed that fruits and vegetables contain small but significant amounts of free galactose. It is estimated that patients on a traditional galactose restricted diets consume greater than 100 mg (Berry et al., 1993) to greater than 500 mg of galactose from fruits and vegetables each day. As a result, British Columbia Children's Hospital's dietitians classified lentils, fruits and vegetables into 3 categories: Acceptable: foods that have free galactose contents that are lower than 10 mg/100 grams fresh weight (gfw) and can be eaten liberally, including asparagus, broccoli, mushrooms and apricot. Use with caution: foods that have moderate amounts of free galactose 10 mg/ lOOgfw to 20 mg/100 gfw and need to be consumed in moderation (no more than 3 times per week) and in smaller portions, including apples, bananas and bell pepper. Questionable: foods that potentially contain  12  large amounts of free galactose (>20 mg/ lOOgfw) and should be avoided from diet including lentils, tomatoes, blueberries, persimmons and papayas. 3.2.3. Limitations of current treatment As mentioned, many foods, especially fruits and vegetables that are usually not considered to contain significant amounts of free galactose have been shown to have levels that may aggravate the chronic complications in galactosemic patients (Berry et al, 1993). Although a number of fruits and vegetables have been analyzed for their free galactose contents (Gross and Acosta, 1991; Gropper et al, 1993; Gropper et al, 2000), some important issues like cultivar differences, storage and processing effects which might cause changes to free galactose levels in foods have not been considered. Moreover, whether the bound galactose in foods can be hydrolyzed and absorbed by enzymes within the human body is not known. Furthermore, an upper limit for intake of free galactose has not been established to help patient and dietitians to monitor galactose intake. With this information, more detailed and thorough recommendations can be given to patients and enabling them to make food choices that could decrease their free galactose intake, and possibly reduce some of the complications of galactosemia.  3.3. Fruit ripening Fruits are generally not harvested at their prime ripened stage, but at a fully developed stage when growth has ceased (Brady, 1987). As soon as fruit is harvested, it is left to survive on its own as the metabolites, being utilized to maintain it's physiological processes, are not replenished any more. Under favourable conditions, softening of fleshy fruits occurs after this stage. Prior to ripening, the fruit has rigid,  13  ordered and well-defined cellular structures, whereas soft and diffused cell walls exist in ripe fruits (Brady, 1987). Softening is an important part of the ripening process in most fruit, and it is widely recognized that changes in cell walls accompany fruit softening (Nunan etal, 1998). 3.3.1. Plant cell wall polysaccharides Three broad classes of polysaccharides are present in the walls of most plant cells: cellulose, hemicellulose and pectin. In fruits and vegetables, the cell wall consists typically of pectic polysaccharides (-35%), cellulose (-30%), hemicelluloses (-25%), and proteins (-10%) (Grant Reid, 1997). Cellulose, in the microfibrillar phase, is a linear polysaccharide chain containing 3000 to 5000 D-glucose residues linked via P-(l—»4) linkages (Grant Reid, 1997). Cellulose self-associates by intermolecular hydrogen bonding to form microfibrils of approximately 30 to 100 such chains and becomes strongly associated with hemicellulose in the cell wall. Extensive intermolecular and intramolecular hydrogen bonds stabilize the composite, giving rise to an amorphous structure (Heredia et al, 1993). Hemicellulose is principally xyloglucan, a linear (3-(l->4) glucosyl chain in which xylose and more complex side chains containing xylose, galactose, and fucose are attached to carbon 6 of glucosyl residues of the glucan backbone at regular intervals (Grant Reid, 1997). In some cases, xylose side chains are attached in a highly regular fashion at three consecutive glucose residues followed by an unsubstituted glucose (Grant Reid, 1997). Pectins function as a hydrating agent and cementing material for the cellulosic network. The highest concentration of pectins in the cell wall is seen in the middle  14  lamella. Pectic substances are a group of complex colloidal plant carbohydrates made up primarily of a-(l,4)-D-polygalacturonic acid and exist in widely varying states of methoxylation and neutralization (Thakur et al, 1997). Pectins are often described in terms of "smooth" and "hairy" blocks which may reside as components of a single pectin polymer (Heredia et al, 1993). The dominant feature of smooth blocks is a linear copolymer of a-(l->4)-linked galacturonic acid and its methyl ester. Inserted within this smooth homogalacturonan polymer are oc-(l->2)linked rhamnosyl residues (Heredia et al, 1993). Rhamnosyl residues within the homogalacturonan backbone serve as attachment sites for arabinose- and galactose-rich side chains (Grant Reid, 1997). "Hairy" pectin blocks are complex heteropolymers with many side chains of neutral sugars (Heredia et al, 1993). The backbone, galacturonic acid and rhamnose, bears numerous side chains rich in arabinose and galactose but also containing other sugars such as fucose, xylose, glucuronic acid and glucose (Grant Reid, 1997). These neutral sugars amount to 10-15% of the pectic weight. The difference between smooth and hairy regions depends on the amount of neutral sugar side chains attached to the galacturonan backbone (Brownleader et al, 1999). 3.3.2. Structural changes of plant cell walls during ripening In most fruits, all the major cell wall polysaccharides appear to be modified during ripening. These events can represent both degradation and synthesis of polymers, and all evidence suggests their metabolism is tightly controlled through regulation of gene expression (Seymour and Gross, 1996). The extent of wall changes differs among fruit types, but most show some of the following modifications.  15  Fruit ripening is frequently accompanied by solubilization of the pectic polysaccharides of the middle lamella, and a loss of some neutral sugars such as galactose and arabinose from the side chains of the polymers (Redgwell et al, 1997; Yoshioka et al, 1994; Gross and Sam, 1984). This can reduce the entanglement of the pectin molecules and increase solubility (Kunzek et al, 1999). Tissue softening during ripening of fruits is generally attributed to enzymatic degradation and solubilization of protopectin. However, reasons for changes during ripening and the effect of the pectolytic enzymes (their accumulation and transformation into active forms) are not sufficiently understood. 3.3.3. Enzymes involved in fruit ripening An increase in water-soluble pectic polysaccharides and the loss of galactose and/ or arabinose from the cell wall occur in many fruits during softening and has been attributed to the action of several enzymes found in fruit tissues (Dick and Labavitch, 1989). However, these enzymes are not the only contributing factors in the modification of pectin solubility. The softening process is complicated by the fact that breakdown or modifications of different components are usually accompanied by the incorporation of newly synthesized components into the wall (Seymour and Gross, 1996). The synthesis of cell wall polymers is probably continuous throughout ripening, and a change in the turnover rate of a particular component will affect the overall wall composition (Brownleader et al, 1999). A number of enzymes including polygalacturonases (PG), pectin methylesterase (PME) and P-galactosidase have been associated with the biochemical processes involved in fruit ripening (Fischer and Bennett, 1991). They induce softening by catalyzing  16  hydrolytic cleavage of unesterified oc-(l->4) galacturonan linkages (Brownleader et al, 1999; Gray et al, 1994), demethylation of the C6 carboxyl group of galacturonsyl residues (Fischer and Bennett, 1991) and removal of galactosyl residues from pectin respectively (Redgwell et al, 1992; Ross et al, 1994). As mentioned, synthesis of modified cell wall polymers could also be involved in cell wall changes resulting in fruit softening. Xyloglucan endo-transglycosylase (XET) is an example of cell wall synthesizing enzyme that rearranges xyloglucan chains from cell walls which might increase the solubility of pectins (Seymour and Gross, 1996).  3.4. Long-term storage of fruits and vegetables The objective of all fruit storage techniques is to delay ripening so that fruit can be marketed at the optimum time without loss of quality. Methods for preserving fresh fruit and vegetables are well developed and rely principally on reduction of the respiration rate of the product by lowering the temperature and by restricting oxygen availability and/ or elevating carbon dioxide concentration in the storage environment (Bishop, 1990). The softening of fleshly fruits during storage is of economic interest, as a process influencing physical damage during handling, disease susceptibility, storage duration and consumer acceptability. Consequently suppression of respiration by refrigeration and/ or by controlled atmosphere may be expected to relate to the duration of storage life that is achievable for any given cultivar (Mathooko, 1996; Johnson and Ridout, 2000; Mahajan, 1994; Massiot et al, 1996).  17  3.4.1. Cold storage in regular atmosphere Storage in a freely ventilated chamber cooled by means of a refrigerating plant is commonly known as cold storage. The principles are simple. Temperature affects all living processes such as growth, ripening, and the progress of rotting. These processes are rapid at high temperatures of 15-21°C and slow at temperatures of 2-7°C. At low temperatures, rotting, which is caused by fungi growing in the tissues, is similarly delayed. The temperature of storage, however, must not be too low. Fruit is killed by freezing and when taken from store becomes brown and soggy and is attacked by molds (Ministry of Agriculture, Fisheries and Food, 1979). Exposure to temperatures approaching their freezing point may also injure some fruits. Chun et al. (1999) demonstrated that galactose and arabinose side chains were still lost from soluble pectin during cold storage with the increase in (3-galactosidase activity in Tsugaru and Fuji apples. In addition, activities of polygalacturonases in Red Delicious apples were shown to be low at the beginning of storage, but they increased with storage time and then declined at the end of storage (Mahajan, 1994; Kovacs et al, 1997b). Therefore, it is evident that enzyme activities are still present at low temperature although at a lower rate than at in higher temperatures. Cold storage in regular atmosphere also has a major influence on the sucrose, glucose, fructose and sorbitol content. A l l sugars decrease over time in cold storage (Drake and Eisele, 1999; Mahajan, 1994). Pectin also declines gradually with the advance in storage period which might be due to conversion of insoluble protopectin into soluble pectin (Mahajan, 1994). The recommended storage temperature for each apple cultivar is the temperature which is most effective in retarding ripening and growth of decay-producing organisms  18  without causing freezing injury. In general, the recommended storage temperature ranges from-1 to 4°C.  3.5. Heat processing The primary object of applying heat to food is to destroy living organisms capable of causing deterioration of the food or endangering the health of the consumers and to eliminate the activities of enzymes that can cause food deterioration. On the other hand, organoleptic and nutritive properties have to be retained to the greatest extent possible. Different severity of heat treatment has different purposes and affects quality and nutrition of food to different degrees. 3.5.1. Blanching Blanching is exposing fruit and vegetable to hot or boiling water or steam, as a pre-treatment to other types of processing. Its main purpose is to inactivate natural food enzymes. It also helps to clean the material and reduce the amount of microorganisms present on the surface, removes gases from the fruit tissues and increases the temperature of the tissue. Some fruits may require blanching prior to filling, particularly if they are to be solid packed, since the softening and shrinkage enables them to be more readily filled into cans. Removal of tissue gases and preheating the product prior to filling are important objectives of precanning operations since they have a great influence on the final level of oxygen in the container and therefore directly influence storage life (Lund, 1975). The advantages of blanching may be slightly offset by the loss of nutritional components that occurs during the operation, so blanching times are kept as short as possible (Burrows, 1996). Nutrient losses caused by blanching result directly from  19  leaching of water-soluble components into the water used during processing. Blanching with steam, hot air or microwaves does not require immersion in water and reduces leaching of these components (Fourie, 1996). 3.5.2. Sterilization A sterile product is one in which no viable microorganisms are present—a viable organism being one that is able to reproduce when expose to conditions optimum for its growth. Temperatures slightly above the maximum for bacterial growth result in the death of vegetative bacterial cells, whereas bacterial spores can survive much higher temperatures. Since bacterial spores are far more heat resistant than are vegetative cells, they are of primary concern in most sterilization processes (Lund, 1975). 3.5.2.1. pH classification of canned foods pH of the food is a critical factor that determines the thermal conditions needed to produce commercial sterility in canned foods. Foods are divided into three categories based upon their acidity and the necessary thermal processing required to effect safety as well as microbiological stability: 1) low-acid foods with pH above 4.6, 2) acid foods with pH values between 3.7 and 4.5 and 3) high acid foods with pH below 3.7. Nonsporeforming and sporeforming aciduric bacteria, yeasts and molds are responsible for the spoilage of acid products (pH below 4.6). In practically all cases, these organisms can be controlled by a short heat process at or below 212°F (Pflug and Esselen, 1979). The heat process is designed to kill the microorganisms that can grow in and spoil the product and not necessarily kill spore-forming organisms such as Clostridium botulinum that will not multiply, and its spores will not germinate at p H below 4.6. The extent of heat process of high acid foods depends on the target organism  20  present that can cause spoilage. For apples, the usual target is Byssochlamys fulva. Ascospores and vegetative cells of Byssochlamys fulva, a heat resistant mold, can survive heat treatments normally applied to canned fruit products and subsequently grow under reduced oxygen, and for this reason, can cause substantial deterioration of canned fruit without recognizable spoilage before the can is opened (Kotzekidou, 1997; Beuchat and Rice, 1979). In high pH (pH 4.6 or higher), low acid canned foods (e.g. canned green beans), the anaerobic conditions that prevail are ideal for growth and toxin production by Clostridium botulinum. This organism is also the most heat-resistant, anaerobic, sporeforming pathogen that can grow in low acid canned foods, therefore its destruction is the criterion for successful that processing of this type of product (Lund, 1975; Hersom and Hulland, 1980). Moreover, destruction of the spores of this organism is generally accepted as the minimum standard of processing for low acid canned foods.  3.5.2.2. D and z values The death of microorganisms at elevated temperatures is generally accepted to be a first order reaction (at constant temperature, the rate of death of the organisms is directly proportional to the concentration present at a particular time). Therefore, there is a defined time during which the number of microorganisms falls to one-tenth of the number at the start of that time interval, irrespective of the actual number. If the surviving fraction is plotted against time, the resulting curve follows a logarithmic course with equal percentages of surviving cells dying in each successive unit of time. The graph obtained by plotting the logarithmic of the number of viable cells against the time  21  of heating at a constant temperature is known as thermal death-rate curve. The slope of the curve determines the Decimal Reduction Time (D), which is defined as the time of heating, in minutes, to reduce the numbers of the survivors to one tenth of the original, i.e. the time for the curve to span one log cycle (Lewis and Heppell, 2000; Hersom and Hulland, 1980). There is a convention that, in low-acid foods, the acceptable level of survival of a spore of C. botulinum is 10~ ; that is 1 spore in 10 initially will survive the 12  12  thermal process. This survival rate of 10" in C. botulinum is known as the 12D concept, 12  which was proposed by Stumbo in 1965. If the D values equivalent to a number of temperatures are plotted on a logarithmic scale against their corresponding temperatures (thermal death time (TDT) curve), a straight line is obtained, and the slope (m) of which determines the z-value where: z = -l/m Therefore, the z-value defines whereby a certain temperature rise will change the decimal reduction time of the microorganisms by a factor of 10, and that this z-value is constant for all temperatures used. 3.5.2.3. Determining lethality of thermal processes It can be observed from the TDT curve or the log survivor plot that the time to achieve a given sterilization varies with temperature. Each time-temperature interval during the heating or cooling of containers has a lethal effect on food spoilage for temperatures above the maximum for growth of the organism. Therefore, it must be possible to add the lethal effect (lethality) at many temperatures in a procedure that will give the sum of the individual effects in terms of minutes at some reference temperature;  22  250°F is the standard reference temperature for low acid food processing (Pflug and Esselen, 1979). The Improved General Method (IGM) is a very common procedure developed for evaluating lethality of thermal processes. It uses the fact that different combinations of time-temperature conditions can achieve the same lethal effect on microorganisms. A reference TDT curve for a hypothetical organism was developed. The z of 18°F was chosen for the reference organism to reflect the temperature sensitivity of C. botulinum; " F ' , the TDT at the reference temperature of 250°F was set arbitrarily to one minute. Lethal rates (L) can be calculated by applying the formula proposed by Ball in 1928 where: L= 1 0 ™ (  z  T= any lethal temperature Tr = 250°F; z = 18°F (in low acid foods) It has been mentioned that lethality is accumulative, therefore: Accumulated lethality = X L * At where At = time interval over which L is constant. Because the lethality of the I G M is based on the unit TDT curve where z= 18°F and the reference temperature = 121°F. It is given the symbol Fo (with units of time) (Lund, 1975; Toledo, 1991). For pasteurization, P value is the equivalent of Fo. However, there is no general guideline for z value and reference temperature for pasteurization processes. The procedure used in applying the I G M requires heat penetration data. Heat penetration profile can be determined by means of inserting thermocouples into cans. This instrument depends on the principle that when two dissimilar metal, such as copper  23  and constantan, are joined at both ends to from a closed circuit and one junction is at a higher temperature than the other, a current is set up and the magnitude of which depends on the temperature difference between the two junctions (Hersom and Hulland, 1980). A potentiometer is put into circuit and as the temperature of the can in the retort is raised, the difference in potential is observed and recorded during the course of retort. Thermocouples are usually mounted in the sides of the test can, the position of the tip being determined by the location of the point of greatest temperature lag. For solid or very viscous packs which heat mainly by conduction, the position of the thermocouple tip is usually taken as the geometric center of the can. For products such as soups or vegetables in liquids, which heat mainly by convection, the thermocouple tip is located on the can axis at a point between the geometric center and the bottom of the can (Pflug and Esselen, 1979; Hersom and Hulland, 1980).  3.5.3. Effects of heat treatment on cell wall polysaccharides During thermal processing, the texture of the product is markedly changed, primarily due to modifications of the coherence of cells and the cell-wall structure. Processing may break glycosidic linkages in the cell wall polysaccharides resulting in an increased amount of soluble fiber (Nyman et al, 1993). Pectin in carrots has been reported to be degraded in heat treatments (Plat et al, 1988). Stolle-Smits et al. (1995) and Massiot et al. (1992) also found the same tendency in processing of green beans. They found that the sugar composition of cell wall polymers in green beans after blanching were not altered and major alterations were observed only during the sterilization of green beans (Stolle-Smits et al, 1995). Besides solubilization of pectins  24  in the "hairy region", they found that the linear homogalacturonan, originating from the middle lamella, is degraded too. In apricots, a high acid food, solubilization of arabinoseand galactose-rich pectic polysaccharides into the canning liquids were also evident (Chitarra et al, 1989). Reinders and Their (1999) showed that besides pectin degradation in canned tomatoes, hemicelluloses were also affected, although to a lesser extent. The degradation of pectin substances mainly proceeds via p-elimination or by hydrolysis of glycosidic bonds and the resulting softening depends considerably on the conditions of the cell's environment (Thakur et al, 1997). In high acid foods, at high temperature, depolymerization by way of hydrolytic cleavage of bonds in the pectin backbone occurs more rapidly. In low acid foods, at high temperature, degradation of pectin polymers by P-elimination is predominant (Voragen et al, 1995; Thakur et al, 1997). The degradation of the pectin molecule in lows acid foods (e.g. vegetables) by Pelimination cleavage of the glycosidic linkage (Kunzek et al, 1999; Voragen et al, 1995) only occurs at glycosidic bonds adjacent to an esterified carboxyl group (Sajjaanantakul et al, 1989 and Thakur et al, 1997). However, to this date, the degradation mechanism of pectic substances in cell walls has not been unambiguously identified due to the complexity of plant tissue and cell wall structure.  3.6. Enzymes used in fruit juice production Fruit juice industry is an area of food technology where enzyme activities and their control dictate the quality characteristics of products. The substrate of interest is pectin and the enzymes of interest are broadly classified as "pectic enzymes". Product  25  characteristics affected by pectin include viscosity, colour stability, clarity and possibly flavour. In the production of fruit juices, there are two possibilities for using enzymes: juice clarification and mash treatment to increase juice yield. 3.6.1. Fruit juice clarification Fruit juice clarification is the oldest and the largest use of pectinases. The raw pressed juice is a viscous liquid due to the presence of a wide range of colliodally dissolved natural polysaccharides and other small particle-like protein fragments or polyphenols (Pilnilk and Voragen, 1993). Addition of pectinase lowers the viscosity and causes cloud particles to aggregate to larger units, which sediment and are removed easily by centrifugation or (ultra)filtration. The cloud particles are defined as a protein nucleus with a positive surface charge coated by negatively charged pectin molecules. Partial hydrolysis of this negatively charged coat leads to the exposure of positively charged surfaces. Thus, the result is a system with particles of unlike charges attracting one another (agglomeration). This results in the formation of a floe which eventually settles out leaving the clear supernatant juice (Kilara and van Buren, 1989; Pilnik and Voragen, 1993; Kilara, 1982). 3.6.2. Enzyme treatments of pulp to increase juice yield (mash treatment and liquefaction) The use of enzymes for apple mash treatment was rather uncommon until the 1970s (Will et al, 2000). There are two main approaches to increase juice yield: to increase the press capacity and total liquefaction of the pulp.  26  3.6.2.1. Increasing press capacity: mash treatment In the late part of the processing season, stored apples are often juiced. The quality of this raw material for juice production is not as good as that of fresh fruit. The longer the storage period, the greater is the change in composition of the apple tissue from protopectin into soluble pectin. The latter leads to increased mash viscosity and impedes the flow of the juice. Therefore, the whole pressing process takes much longer. Enzymatic hydrolysis of the soluble pectin by means of a special mash enzyme removes these drawbacks and provides a better yield. The quantity of juice can be increased significantly. Enzyme preparations performing well in juice clarification are also suitable for the enzyme treatment of pulp (Pilnik and Voragen, 1993). Commercial pectin enzymes (pectinases) generally contain mixtures of enzymes with combined effects of pectinesterase (PE), and depolymerases (polygalacturonase (PG), and pectintranseliminase (PTE)). Almost all the commercial pectinase products contain the three single activities in different ratios, which depend mainly on the production strain and the production technology of the enzyme manufacturer (Ceci and Lozano, 1998). Pectins are first de-esterified by P E prior to conversion to low-esterified pectin or polygalacturonic acid. Scission within the polygalacturonic acid chain is affected by depolymerases in two different ways: by hydrolytic reaction by P G and by highly specific transeliminative cleavage. The latter class of depolymerases are called the endopectin lyases or PTE are important constituents of pectinolytic enzyme products. Hydrolysis of pectins occurs mostly through the action of PG, either endo- or exo-PGs, depending on the type of cleavage (Zadernowski, 1999).  27  Modern apple mash enzymes only break down dissolved pectin in the juice. This results in a viscosity reduction and enables the juice to run off more easily and quickly (Ibarz et al, 1997). At the same time, a macerating effect on undissolved protopectin is avoided, which would otherwise destroy the mash structure and promote the development of fine suspended matter (Urlaub, 1996). 3.6.2.2. Liquefaction Enzymes can also be added to liquefy the pulp so that pressing is not necessary. Modern commercial methods of juice manufacture routinely use proprietary cocktails of carbohydrase enzymes to degrade both the pectinaceous middle lamella and the cell wall to provide increased yields and release of juice (Beveridge and Weintraub, 1997). In addition to the pectinases, liquefacation enzyme preparations have secondary, macerating enzymatic activities of cellulases, hemicellulases, xyloglyconases, arabanases and galactanases. They not only degrade soluble pectins but also protopectins and other cellular structures like cellulose and hemicellulose (Zadernowski, 1999). The viscosity of apple pulp decreases during treatment with pectinases, cellulases (macerases) and a mixture of the two enzyme preparations. When they are mixed, a synergistic effect is seen (Will et al, 2000; Hbhn, 1996). At this stage, viscosity reaches complete liquefaction and cell walls disappear under microscopic examination. Using special liquefaction enzymes has several aims: 1) Decrease mash viscosity—the lower the mash viscosity, the more effective is the separation of the juice from the solids. Hydrolyzing the soluble pectin leads to a considerable drop of the mash viscosity. Although the principle is the same as in  28  conventional mash treatment, it has to be kept in mind that, in mash liquefaction, the viscosity drop is much bigger. 2) Increase in Brix values—additional enzyme activities like cellulase and hemicellulases lead to an increased extraction of soluble solids (expressed by Brix values), thus improving the total yield of juice. The liquefied juices are cloudy for apple pulp. Then, the liquefacation products can be clarified further by usual techniques (Hohn, 1996). Total enzymatic liquefaction, as opposed to normal apple mash treatment, calls for pectinases that hydrolyze both dissolved pectin and undissolved protopectin and macerases that can degrade other cell wall materials. During total liquefaction, the apple primary cell walls and middle lamella are attacked. Released cell wall material gets into the extraction juices in the form of poly-, oligo-, or monosaccharides (Will et al, 2000; Beveridge, 1997).  3.7. Apples Apple (Malus domestica Borkh.) is a fruit that can be stored, canned and juiced. Due to it's once a year harvest in the fall, apples are stored to reduce rate of ripening to make them available during other times of the year. Apples are also canned for use generally in pie fillings and apple juice is the second most popular juice in North America. As a result, apple is a very good model for studying the influence of these processing techniques on free galactose concentrations for this research.  29  3.7.1. A p p l e p r o d u c t i o n  Apple is a highly remunerative and delicious fruit, grown in temperate regions. It is the second largest fruit crop produced averaging around 39,404,000 metric tons a year (FAO, 1995). Canada produced about 511,485 tons of apples in 2001 and British Columbia produced about 125,650 tons which is about 25% of the total Canadian production (Statistics Canada, 2002). 3.7.2. Composition  Carbohydrates are the principal food constituents in apple, with starch and sugars being the available carbohydrates and pectin, cellulose and hemicellulose the unavailable fractions. Total carbohydrates in fresh apples accounts for about 15%, comprising 0.895.58% each of fructose and glucose; and 0.88-5.62% sucrose. Pectin accounts for 0.51.6% (Thakur et al., 1997). Gross and Acosta (1991) reported that apples have 8.3mg/100g fresh weight of free galactose, however, they did not specify what variety was evaluated. It is important to specify the cultivar because different cultivars of apples may have different sugar composition. This is supported by the fact that Fuleki et al. (1994) and Fourie et al. (1991) found that varietal differences affect the amount and composition of sugars in apples, for example: 5.48g fructose/lOOg was found in Starking apples whereas only 3.81g fructose/ lOOg was found in Granny Smith apples. 3.7.3. Storage  Apples are harvested in the fall. In order for apples to be available all year round, they are stored at low temperature or in controlled atmosphere environments to delay ripening. There are innate differences in the quality of apples of different cultivars that characterize their potential for long-term storage. Apples of different cultivars vary in  30  their potential to store and in their response to methods used to prolong storage life (Johnson and Ridout, 2000). This cultivar-storage environment interaction is particularly important since they determine tolerances to the stresses that are imposed by low storage temperatures and by low oxygen and elevated carbon dioxide concentrations in the storage environment. 3.7.4. Processing Excess apples that are not stored or have been in storage for some time are processed routinely. Approximately 46% of the apples produced in the North America receive some type processing and are made into five main products: juice, canned sauce, canned slices, dried apples and frozen slices. Apple juice and canned sauce / slices are the dominant products, accounting for about one-half and one-third, respectively, of all processed apple products. A few apples are also processed into vinegar, jelly, apple butter, mincemeat, and fresh slices. Furthermore, small quantities are also made into apple wine, apple essence, baked whole apples, apple rings, and apple nectar (Way and McLellan, 1989).  3.7.4.1. Canning Solid pack is by far the most common way of canning apples. The apples are blanched by passing the sections through a steam tunnel or by immersion in hot water for a sufficient time to render the slices pliable. Blanching of apples is essential to remove oxygen from the tissues and thus prevent pinholing in the cans during storage (Lal Kaushal and Sharma, 1995). Filling should be carried out immediately to prevent any appreciable drop in temperature. Ideally the temperature should be above 70°C. The  31  cans are then topped up with boiling water, taking care to maintain the headspace at between 8mm to 10mm. After filling, the cans should be thoroughly exhausted to ensure that all air is removed. When the cans have been seamed, they are heat treated in a retort for sufficient time to raise the can center temperature (Burrows, 1996). 3.7.4.2. Juice production Another popular apple product is apple juice. The greatest volume of apple juice sold in North America is in the form of clarified apple juice. The totally clear and shining characteristics of clarified apple juice are preferred by consumers. The production of clear apple juice requires the removal of suspended material and prevention of the development of turbidity after juice bottling. Freshly pressed juice contains suspended solids that are deliberately precipitated prior to filtration. This precipitation step is called clarification. Preparation of this product involves pressing the apples, clarification treatment and filtration (Kilara and van Buren, 1989). Enzymatic preparations can be added during pressing to increase yield and/ or clarification steps to help produce clear juice.  3.8. Carbohydrate analysis 3.8.1. Extraction of sugars The first step in the analysis of the free sugars depends primarily on whether the sugars are in solution or whether they need to be extracted from the foodstuff. Free sugars are extremely soluble in aqueous solutions which makes water the obvious choice as extracting media (Southgate, 1976). However, aqueous solutions also extract many other polar substances, e.g. amino acids and proteins, that interfere with the subsequent  32  measurements of sugars, and so quite extensive clean-up procedures are frequently necessary before measurements can be made. The extraction solvent must therefore be chosen to have adequate solubilizing effect on the components of interest (sugars) but not on potential interferents (MacRae, 1983). The free sugars are generally soluble to a significant extent in aqueous alcoholic solutions and, as proteins and virtually all polysaccharides are insoluble at alcoholic strengths above 70-75% (v/v), these reagents have formed the basis of most extraction procedures in unified analytical methods. Several different techniques for extraction with aqueous alcohol have been employed and ethanol, methanol and iso-propanol have been used extensively. The most commonly used extraction solvent is 80% ethanol (AOAC Method 922.02, 925.05). Since much of the composition of a food (other than water) is in the form of polymers, and almost all polysaccharides and proteins are insoluble in 80% ethanol, this extraction solvent is rather specific. This solvent system provides a good compromise between efficiency and selectivity of extraction (BeMiller and Low, 1998).  3.8.2. Quantification of sugar mixtures Extracts of foods that contain a single species of sugar are relatively rare in practice. The measurement of free sugars in most foodstuffs therefore involves the analysis of mixtures. The available assay methods for monosaccharides include chemical, colorimetric, chromatographic, electrophoretic, optical and biochemical procedures (Pomeranz, 1987). The general lack of real specificity in the analytical  33  methods for sugars, with the exception of the biochemical procedures, implies that accurate analysis of mixtures must be preceded by separation. 3.8.3. Chromatography The separation and quantitation of the components in mixtures of monosaccharides forms an important part of carbohydrate analysis. There are two main methods available, high performance liquid chromatography (HPLC) and gas chromatography (GC). As a broad generalization, H P L C is particularly suitable for rapid analyses of large sample quantities of simple mixtures of monosaccharide and oligosaccharide where ease of sample preparation (no derivatization required) and speed of analysis are advantageous. Analysis times are generally shorter for H P L C . G C is generally favored for complex samples or for those containing unknown or low concentrations of carbohydrates. Sample preparation is required to extract and purify the desired carbohydrates. Moreover, as carbohydrates are nonvolatile due to the presence of highly polar hydroxyl groups, they have to be converted to volatile derivatives before submitting to GC analysis. Although more tedious to perform, G C using a flame ionization detector is typically a factor of 10 times more sensitive than H P L C using refractive index detection (Folkes, 1983; Bradbury, 1990), therefore suitable for analysis of carbohydrates that are present in low amounts. Although G C and H P L C analyses are suitable for different types and mixes of sugars, there are protocols available for separating and analyzing most mixtures of carbohydrates by either method (Chaplin, 1986).  34  3.8.4. Gas chromatography GC depends not only upon adsorption, partition, and/ or size exclusion for separation which H P L C uses for carbohydrate analysis, but also upon solute boiling point for additional resolving powers. Thus, the separations accomplished are based on several properties of the solutes (Belanger et al, 1997a). This gives G C virtually unequal resolution powers as compared to most other types of chromatography. 3.8.4.1. Principles The basis for gas chromatographic separation is the distribution of an analyte between two phases—mobile phase and stationary phase. The mobile phase is usually an inert gas which moves through a GC column and passing over the stationary phase. The stationary phase is typically a liquid coated directly on the inside walls of a tube (Reineccius, 1998; Scott, 1998). A sample is introduced into the mobile phase through an injection system. Once the sample enters into contact with the mobile phase in the column, the components of the sample interact to varying extent with the stationary phase and partition between the stationary and the mobile phase, resulting in differential migration rates through the column (Wang and Pare, 1997). Equilibrium occurs between the gas and the stationary phase when the probability of a solute molecule striking the surface and entering the stationary phase is the same as the probability of a solute molecule randomly acquiring sufficient kinetic energy to leave the stationary phase and enter the gas phase. At all times, the distribution system is thermodynamically driven toward equilibrium. However, since the mobile phase is constantly moving, it will continuously displace the concentration profile of the solute in the mobile phase forward, relative to that in the  35  stationary phase. As a result of this displacement, the concentration of solute in the mobile phase at the front of the peak exceeds the equilibrium concentration with respect to that in the stationary phase. It follows that solute from the mobile phase in the front part of the peak is continually entering the stationary phase to reestablish equilibrium as the peak progresses along the column. At the rear of the peak, the converse occurs. As the concentration profile moves forward, the concentration of solute in the stationary phase at the rear of the peak is now in excess of the equilibrium concentration. Therefore, solute leaves the stationary phase and enters the mobile phase in an attempt to reestablish equilibrium. The solute band moves through the chromatographic system as a result of the solute entering the mobile phase at the rear of the peak and returning to the stationary phase at the front of the peak. At any given time, a particular analyte is either in the mobile phase, moving along with the moving gas, or in the stationary phase and diffusing in it. Such sorption-desoprtion process occurs repeatedly until each analyte forms a separate band and leaves the column (Wang and Pare, 1997; Reineccius, 1998). As the analyte emerges from the end of the GC column, it enters into a detector and produces some form of signal, the strength and duration of which is related to the amount of, or to the nature of the analyte. Generally, the signal is amplified and passed to an electronic integrator, a computer, a strip chart recorder or other means by which the chromatogram is obtained and the quantitation of the analyte is then made (Scott, 1998). 3.8.4.2. Sample preparation—derivatization The compounds must be thermally stable under G C conditions in order to be determined by GC. For some compounds (e.g. aroma compounds) the analyst can simply isolate the components of interest from a food and directly inject them into the GC. For  36  compounds that are thermally unstable or too low in volatility (e.g. sugars), a derivatization step must be carried out prior to G C analysis (Reineccius, 1998). Carbohydrates are non-volatile and in order to make them suitable for analysis by GC, it is necessary to form chemical derivatives that have enhanced volatility. This is usually done by applying derivatization procedures that convert the hydroxyl groups of the carbohydrate molecules to ether (e.g. silyl, methyl) or ester groups (e.g. acetyl, trifluoroacetyl). The most widely used derivatives are the trimethylsilyl (TMS) ethers (Bradbury, 1990) due to the simplicity of the derivatization procedure. For more complex mixtures, a modified derivatization procedure (e.g. TMS-oxime) is usually more appropriate. This is because most carbohydrates exist in solution as a mixture of anomeric and acyclic forms, and direct ether formation or esterification leads to a number of stereochemical isomers, each of which gives a peak on the G C chromatogram. This causes problems in resolution as each reducing sugar produces two to four peaks (Reineccius, 1998; Wang and Pare, 1997). In order to reduce the number of isomers produced, an extra derivatization step, involving modification of the reducing function at the anomeric carbon, is introduced. Conversion to oximes and methyloximes are the modifications that are preferred. Oxime and methyloxime derivatives exist in two closely related syn- and anri-isomeric forms. It is worth noting that non-reducing carbohydrates are not converted to oximes, so samples that contain non-reducing and reducing carbohydrates are actually analyzed as a mixture of TMS and TMS-oxime derivatives (Bradbury, 1990: Folkes, 1983). Another common derivative used is alditol acetates. These derivatives are widely used for monosaccharide analysis. A major advantage of this method in comparison to  37  TMS derivatization is that a single peak is produced for each monosaccharide which is readily separated on columns of medium to high polarity. Moreover, once formed, the alditol acetates are extremely stable, thus allowing post-derivatization clean up and treated samples can be stored at -18°C for extended periods (Fox et al, 1989). However, this derivatization method only can produce derivatives for reducing monosaccharides. Fructose is reduced to a mixture of D-glucitol (sorbitol) and D-mannitol (BeMiller and Low, 1998). There are two preparation steps involved: reduction of aldehyde groups to primary alcohol groups with an excess of sodium borohydride dissolved in a solution of dimethyl sulphoxide with methylimidazole as a catalyst and conversion of the reduced sugars into volatile acetates with acetic anhydride (Bradbury, 1990; BeMiller and Low, 1998; Chaplin, 1986; Southgate, 1976; Folkes, 1983). 3.8.4.3. Columns There are two general classifications of gas chromatographic columns, packed and capillary or open tubular columns. They differ in that the carrier gas must percolate through a porous bed in a packed column but in the capillary column the gas flows through a central aperture that is unimpeded throughout the entire length of the column. Therefore, the flow impedance of the packed column is much greater than that of the capillary column when operated at the same gas velocity. For this reason, capillary columns can be made much longer and thus produce many more theoretical plates than the packed column (Scott, 1998). Capillary columns can subdivided into wall-coated open tubular (WCOT) type and porous-layer open tubular (PLOT) type. The most important and the most widely used type is WCOT column. If not specified, the capillary column is generally of the  38  W C O T type. The internal surface of the capillary column is coated directly with a thin film of liquid. Capillary columns vary in length from 25m to 100m and in internal diameter from 0.05mm to 0.53mm (Wang and Pare, 1997). Both packed and capillary columns are used for the analysis of food carbohydrates. For relatively simple analysis of the common sugars, a short packed column will suffice. However, more complex mixtures, or samples containing interfering components, will often require the higher resolution provided by the capillary columns. A variety of stationary phases are available, ranging from the lower polarity methyl silicone gums, which are suitable for the separation of TMS derivatives, to the higher polarity phases such as cyanopropylphenylated silicones, which can be used for acetylated and trifluoroacetylated derivatives. The former phase types are more thermally stable and can be operated at higher temperatures, thereby speeding analysis (Bradbury, 1990; Scott, 1998; Wang and Pare, 1997). In general, non-polar stationary phases separate mixtures of non-polar components mainly on the basis of boiling points, with dipole-induced dipole interactions being significant for polar solutes. Polar stationary phases have specific dipole-dipole, van der Waals and hydrogen-bonding interactions with solutes (Gordon, 1990).  3.8.4.4. Detectors The G C detector senses the emergence of an analyte as it exits from the G C columns, producing an electrical signal that is proportional in intensity to the concentration or the mass of the eluted analyte. The most favoured detector for carbohydrates is the flame ionization detector (FID), which allows high-sensitivity  39  detection down to the nanogram level. For organic substances, the response is approximately proportional to the molecular weight and thus introduction of the bulky trimethylsilyl group, which triples the molecular weight of a typical carbohydrate, gives high sensitivity (Bradbury, 1990). The FED responds to the mass of eluent per unit time (mass flow). A flame is produced at the tip of the jet of the FID by combustion of the mixture of hydrogen and air. When carrier gas is eluting from the column, there is virtually no signal and a stable baseline is achieved, but when organic compounds elute from the column, they burn in the flame to form radicals which would suffer some ionization. The ions will be attracted by the FED collector on which a polarizing voltage is applied, and produce a current, which is proportional to the quantity of analyte in the flame. For optimal operation, the carrier, hydrogen and airflow must be properly set and adjusted (Gordon, 1990; Wang and Pare, 1997).  40  4. Materials and Methods  4.1. Storage trial 4.1.1. Produce selection Four varieties of apples were obtained from Dr. Sam Lau of the Pacific Agri-Food Research Center orchards in Summerland. The four cultivars are Braeburn, Spartan, Fuji and Red Delicious. They were all harvested in October 2000, at optimum maturity. A l l apples were obtained from a research station instead of the market place, to make certain that apples were from the same cultivar and orchard, in addition, harvested at the same time, ensuring all fruits had the same biological age. 4.1.2. Storage condition Apples were stored in fiber board cartons at 4°C in a walk-in chamber at regular atmosphere immediately after harvest at Summerland. After all cultivars were harvested, they were transported to U B C on 25 October, 2000. Immediately after arriving at the th  laboratory at U B C , the apples were placed in storage in a walk-in cold chamber. Each cultivar was stored in separate fiber board cartons at 4°C in normal atmosphere. The fruits were examined periodically throughout the storage period to ensure that there was no moisture buildup on fruit surfaces and bruised, infected apples were removed. 4.1.3. Sample selection Sampling (time 0) began immediately on 26 October, 2000 after the apples were th  collected from the Research Station at Summerland. Sampling was carried out during a 9-months storage period from October 2000 to July 2001. Samples were taken 4 times during the storage trial on the 26 of October and subsequently at 3 months intervals. At th  41  each sampling period, 4-5 apples from each cultivar were randomly removed from storage. Fruits were examined visually for uniformity in shape and firmness, and free from defects like brown core, senescent breakdown, bruising and infections. Defected fruits were rejected and replaced with other fruits from storage.  4.2. Thermal processing Experiments were conducted to establish the effect of thermal processing on the level of free galactose. Samples included Fuji apple, as an example of high acid/ low pH product that is typically pasteurized, and green beans as an example of low acid/ high pH product that is typically processed to achieve commercial sterility. Fuji apples were from Pacific Agri-Food Research Center orchards and they had been stored for 9 months before processing. Green beans (P. vulgaris L.) were bought from a local market place in October, 2001. The heat treatments analyzed were 1) blanching, 2) blanching and pasteurization, 3) blanching and double pasteurization, 4) blanching and canning to commercial sterility and 5) blanching and canning to twice the commercial sterility. Pasteurization and canning were carried out using the batch-style steam retort in the Department of Food, Nutrition and Health, U B C .  4.2.1. Blanching Fuji apples were peeled, cored and sliced into 0.5 cm wide strips with a household food slicer and blanched in boiling water in a steam kettle for 2 minutes. Green beans were cut into parts of 2.5 cm in length and blanched in boiling water in a steam kettle for 4 minutes. Three samples were taken from each blanching session, and 3 blanching  42  sessions were done. The blanched samples were stored at 4°C until sugar extractions were carried out. The rest of the blanched produce was used for canning. 4.2.2. Canning 4.2.2.1. Canning of apples After blanching, the apple strips were drained with a household sieve and 330 g of apple strips were hand-filled into a No.2 (307x 409) " C " enamel can (Wells Can, Burnaby, BC). Blanching water was then used to fill the cans, leaving 10mm headspace and the cans were sealed immediately with a hand operated can sealer. The steam retort was turned on as soon as the cold spot in the can cooled to 160°F. After the retort reached 212°F, the cans were kept at 212°F for 20 minutes to achieve commercial pasteurization conditions and 38 minutes to achieve twice the commercial pasteurization conditions. 4.2.2.2. Canning of green beans The green bean strips were drained with a household sieve and 350 g of green bean strips were hand-filled into a No.2 (307x 409) " C " enamel can (Wells Can, Burnaby, BC), after blanching. Blanching water was then used to fill up the cans, leaving 10 mm headspace and the cans were sealed immediately with a hand operated can sealer. The steam retort was turned on as soon as the cold spot of the product cooled to 120°F. Once the retort temperature stabilized at 250°F, the cans were held for 11 minutes to achieve commercial sterility and 16 minutes to achieve twice the commercial sterility at the retort temperature.  43  4.2.2.3. Heat penetration determination at cold spot during processing Six cans were canned in each canning session for each product and triplicate canning sessions for each product were done. Temperature measurement at the cold spot was carried out using Ecklund needle-type rigid thermocouples. The thermocouples were inserted 1 inch from the bottom of the can (cold spot); locking receptacles and connectors were used to hold the thermocouple in place. Three cans were processed together with other cans during each processing run, to monitor the heat penetration (temperature) at the cold spot. Thermocouples were connected to a data collecting system (Data Taker DT 100F, Data Electronics (Australia) Pty. Ltd.) and temperature changes (at the cold spot inside the can and retort temperature) were recorded with Decipher version 1.02 (Data Electronics (Australia) Pty. Ltd.). The changes in temperature of the cold spot recorded during thermal processing were used to calculate the P or F values using the 0  Improved General Method (IGM). The equation used was: Total lethality= Z l O ~ * At T= temperature of the cold spot in can (°F) T = reference temperature (°F) (T  Tr)/z  r  At = time interval that the temperature was taken (minutes) T of 212°F and z of 16°F were used to calculate the total lethality of canned apple slices. r  T of 250°F and z of 18°F were used to calculate the total lethality of canned green beans. r  4.3. Juice production Information on how different enzyme preparations affect free galactose concentration in juice products was also obtained. Red Delicious apples from Okanagan, B C , were bought from a local market place in October, 2001. Triplicates of each type of juice were made. In each session, about 10 Red Delicious apples were peeled and pureed  44  with a food processor. The apple puree then underwent different treatments to produce four types of apple juice as described below. Treatment conditions and concentrations for enzymatic preparations were followed according to manufacturer's recommendations. A l l juices produced were stored at -25°C until sugar analysis was carried out. 4.3.1. Juice 1: Control juice (no enzyme preparation added) A 400 g sample of apple puree was spread evenly on a sieve (mesh No. 100). The puree was set aside at room temperature for 30 minutes and the juice was collected in a bowl underneath the sieve. The juice was then centrifuged at 16300x g for 15 minutes at 20°C, followed by pasteurization by placing juice stored in a 250 mL beaker in a boiling water bath until the juice reached 95°C for 1 minute. 4.3.2. Juice 2: Juice with liquefaction enzymes added Pectinex Ultra-SPL (0.044% (v/ w)) (Nova Industries AS, Copenhagen, Denmark) was added to 400 g of apple puree. The puree was heated to and held at 50(no enzyme preparation added) for 2 hours with constant stirring. The puree was then spread evenly on a mesh sieve (mesh No. 100). The puree again was set aside at room temperature for 30 minutes and the juice extracted by gravity from the sieve was collected in a bowl set underneath the sieve. The juice was then centrifuged and pasteurized as stated in Section 4.3.1.. 4.3.3. Juice 3: Juice with liquefying enzymes and clarification enzymes added To the juice produced in section 4.3.2.,100 mg/ L Ultrazym 100 (Nova Industries AS, Copenhagenm Denmark) was added. Then the juice was heated to and held at 40°C for 20 minutes with constant stirring. Afterwards, the resulting juice was centrifuged and pasteurized according to the procedures stated in Section 4.3.1..  45  4.3.4. Juice 4: Juice with clarification enzymes added After control juice (Section 4.3.1.) was made, 100 mg/ L of Ultrazym 100 (Nova Industries A S , Copenhagenm Denmark). The juice was heated to and held at 40°C for 20 minutes with constant stirring. The resulting juice was clarified with centrifugation and pasteurized following the procedures in Section 4.3.1.. 4.3.5. Brix and pH determination The solids content was determined as °Brix with a bench refractometer at 20°C. pH was determined by using a bench top pH meter (Fischer Scientific) at 20°C. Triplicate samples were analyzed for each apple juice.  4.4. Sugar extraction Free galactose present in food materials was extracted using established protocols (Gross and Acosta, 1991; Gross et ah, 1995). Fresh apples and green beans were prepared as for household consumption. The most commonly eaten portion of fresh apple was taken from at least 4-5 samples, peeled, cored with a peeler knife and diced into approximately 0.5-1 cm sections and mixed. Ten to fifteen stalks of fresh green 3  beans were cut into parts of 2.5 cm in length and mixed, triplicate samples were taken from each produce. . Triplicate samples from each processing session were also taken from blanched, commercial pasteurized/ sterilized and double commercial pasteurized/ sterilized apples and beans. Sugars were immediately extracted from fresh and blanched samples; canned products were stored at 4°C for 2 weeks-prior to sugar extraction. Blanched and canned  46  apple slices and green beans were drained to remove excess processing liquids with a household sieve for 30 minutes before samples were drawn. Three 10 grams fresh weight samples were taken and each was placed in 30 mL of 80% ethanol. Each sample was then placed in boiling water bath for 10 minutes and stored at -18°C for 16 hours. The samples were then homogenized with a Polytron (Brinkmann Instruments) at speed 7. The homogenates were vacuum filtered through Whatman No. 1 filter paper. Residues were rinsed with 3 mL of 80% ethanol and the filtrates were combined. Then the filtrates were centrifuged at 20,000x g for 15 minutes. Supernatants were collected and brought up to 30 mL total volume with 80% ethanol. A 10 mL aliquot of each sample was then passed through a Cis Sep-Pak cartridge (Waters Corporation), the cartridge was then rinsed with 2 mL water and the filtrates were pooled. Three 3 mL (for alditol acetate derivatization) and 0.3 mL (for TMS-oxime derivatization) aliquots from each extract were taken to dryness in a vial with a stream of nitrogen at 45°C. Dried samples were stored at -25°C until derivatization for gas chromatography was carried out. Extraction of sugars was unnecessary for apple juice; therefore 0.3 m L of each type of apple juice was used directly for analysis. Before derivatization, the moisture in each sample was evaporated using SCI 10 SpeedVac concentrator (Savant Instruments, Inc.) at low drying rate.  47  4.5. Sugars derivatization and quantification Two sugar derivatization methods were evaluated. TMS-oxime derivatization was the method of choice (refer to discussion section 5.1. for explanation) and was used in the analysis of all extracted sugars from different treatments. 4.5.1. Alditol acetate derivatization 4.5.1.1. Derivatization procedures Soluble monosaccharides were derivatized into alditol acetate derivatives according to the protocols established by Harris et al. (1988) and Blakeney et al. (1983). This derivatization method involves 2 steps: reduction and acetylation. 1) Reduction Reduction was carried out with a solution of sodium borohydride (Sigma Chemicals) in dimethyl sulphoxide (Sigma Chemicals) prepared by dissolving 2 grams of sodium borohydride in 100 mL dimethyl sulphoxide at 100°C. The extracted monosaccharides were dissolved in 0.1 mL 0.1 M sodium hydroxide (Fischer Scientific) and 1 mL sodium borohydride solution was added. The solution was then placed in a 40°C water bath for 90 minutes. After reduction, the excess of sodium borohydride was decomposed by the addition of 0.1 mL 18 M acetic acid (Fischer Scientific) and the solution was cooled to room temperature before proceeding to the next step. 2) Acetylation 1-Methylimidazole (0.2 mL) (Sigma Chemicals) and 2 mL acetic anhydride (Sigma Chemicals) were added to the reduced monosaccharides and the components were mixed. The solution was left, to stand at room temperature for 10 minutes. Distilled, deionized, water (5 mL) was added to decompose the excess of acetic  48  anhydride afterwards. When cooled, 1 mL dichloromethane (Fischer Scientific) was added and the mixture mixed with a vortex mixer. After phases had separated, the lower phase was removed and transferred to 4 mL glass vial (Supleco Inc.) and dried with a stream of nitrogen at 45°C. Fresh dichloromethane (1 mL) was then added to each vial and the vials were stored at -25°C until gas chromatography was carried out. If this phase was cloudy, a little anhydrous sodium sulphate was added to absorb the moisture. 4.5.1.2. Gas chromatography for alditol acetate derivatives Complete separation of galactose from other soluble sugars was accomplished using capillary gas chromatography. Detection and quantification of alditol acetate derivatives were performed using a gas chromatogram (Varian Associates, Inc., Palo Alto, C A , U.S.A.) equipped with a flame ionization detector. Chromatography was performed on a SP-1225 (medium polarity) column (60m x 0.25 mm; 0.25 u.m flim thickness, Supelco Inc., Toronto, O N , Canada) under the following conditions: carrier gas, helium; flow rate, 1.7 mL/ min; injection volume 1 uL. The oven temperature program consisted of an initial temperature of 220°C held for 10 min, then increasing to 240°C at 5°C/ min, and held for 40 min. 4.5.2. Trimethylsilyl (TMS) derivatization 4.5.2.1. Derivatization procedures Sugars extracted from fruits and vegetables were derivatized into trimethylsilyether/ester (TMS) and TMS-oxime derivatives according to the protocols established by L i and Andrews (1986), Al-Hazmi et al. (1985), Long and Chism UI (1987) and Katona, et al. (1999).  49  The sugars and 1 mL pyridine (Sigma Chemicals) (containing 1.25 g hydroxylamine hydrochloride 100 mL  _ 1  (Sigma Chemicals) and 0.1 mg/ mL phenyl-(3-D-  glucopyranoside (internal standard)), were mixed and heated for 30 minutes at 75°C. The cooled samples were then trimethylsilylated with a mixture of 1.8 mL hexamethyldisilazane (HMDS) (Sigma Chemicals) and 0.2 mL trifluoroacetic acid (TFAA) (Sigma Chemicals) for 60 minutes at 100°C. Thereafter, the solutions were transferred to 4 mL glass vials and were evaporated to dryness under nitrogen at 45°C. One mL of a solution of H M D S : T F A A = 9:1 was then added to each vial. 4.5.2.2. Gas Chromatography for T M S - o x i m e derivatives  Complete separation of galactose from other soluble sugars was accomplished using capillary gas chromatography. Detection and quantification of galactose T M S oxime derivatives was performed using a gas chromatogram (Varian Associates, Inc., Palo Alto, C A , U.S.A.) equipped with a flame ionization detector. The TMS and TMS-oxime derivatives were separated by capillary gas chromatography with a non-polar column (OV-1) (30 m x 0.25 mm i.d., 0.25 p,m flim thickness, Supelco Inc., Toronto, ON, Canada) under following conditions: carrier gas, Helium; flow rate, 1 mL/ min; injector port temperature, 250°C; detector temperature, 300°C; injection volume 1 |xL. The oven temperature program consisted of an initial temperature of 180°C held for 5 minutes, that was increased to 200°C at 5°C/ minute, held for 11 minutes and increased to 270 °C at 10 °CJ minutes and held at 270 °C for 13 minutes. The total analysis time for each injection was 40 minutes. Flow rates of the helium make-up gas and the hydrogen gas were set at 30 mL/ min and for air at 60 mL/ min. The head pressure of the column was set at 15 psi, and the flow rate of the helium  50  carrier gas was 1.7 mL /min. Peak integration was performed with the JCL 6000 Chromatography Data System for PC (Jones Chromatography, Lakewood, CO, U.S.A.). A 10-point standard curve using galactose standards with concentrations from 0.001 mg/ mL to 0.06 mg/ mL and internal standard (phenyl-(3-D-glucopyranoside) concentration of 0.1 mg/ mL was performed to determine the linear range and response factor of galactose peak area with reference to the peak area of the internal standard. The galactose extraction and quantitation procedures were validated by comparing the amount of galactose in an apple sample that had no galactose added with an identical sample that had been spiked with galactose equivalent to 5, 10 and 20 mg/ 100 gfw. Samples were run in triplicates.  4.6. Statistical analysis A l l data collected from this research was statistically analyzed using Minitab statistical software (Minitab release 13.30, Minitab Inc., PA). Two-way A N O V A with replication was used for the analysis of the effect of storage and cultivar differences on the free galactose content of apples. One-way A N O V A , Tukey's multiple comparison test and two-way A N O V A were used to analyze the effect of heat treatment on apples and green beans and effect of different enzymatic preparation on amounts of free galactose in clear apple juice. A l l treatments were considered to be significantly different at p< 0.05. Due to problems in sampling in the storage study (Section 5.1.3.), one-way A N O V A , Tukey's multiple comparison tests and two-way A N O V A were done with 3 readings (n=3) (consisting of the mean ±2.1 times the standard deviation obtained from  51  the individual apple storage study) for each data point, as a conservative estimate of the highest possible variation that might occur from the sampling method. Treatments were considered to be significantly different from each other at p< 0.05.  52  5. Results and Discussion  5.1. Derivatization methods  Derivatization of sugars into alditol acetate derivatives and trimethylsilylatedoximes (TMS-oxime) derivatives were evaluated in this research. They represent two classes of derivatives that are very commonly used and can provide satisfactory separation of sugar mixtures. Alditol acetate derivatization method produces a single derivative from each carbohydrate while the TMS-oxime derivatization method produces 2 peaks for each carbohydrate. 5.1.1.  A l d i t o l acetates derivatization  The alditol acetate derivatization method was thought to be more advantageous than the TMS-oxime derivatization method because only one peak is produced for each monosaccharide; therefore identification of peaks is easier and sensitivity may be greater than with the TMS-oximes derivatization method (Chaplin, 1986, Blakeney et al, 1983). Moreover, it was used in previous studies (Gross and Acosta, 1991; Gropper et al, 1993; Gross et al, 1995; Gropper et al, 2000) where free galactose contents in fruits and vegetables were evaluated. Figure 2 showed an example of the gas chromatogram of a sugar mixture of galactose (0.1 mg/ mL) and the internal standard, allose (0.1 mg/ mL), illustrating the single peaks produced for both galactose and allose derivatives. A 9-point standard curve was produced with galactose concentration ranging from 0.01 mg/ mL to 0.4 mg/ mL (Figure 3). Within this range, a linear response up to the tested galactose concentration with a response factor of 0.8914 and r of 0.9952 was obtained. However, when 2  53  m co  13 a cu If)  te  11  U V  "2 8 CM  •S 1  s  o  <u II ce CS O cu c  E  « *  CM O 0> t-  ^ « CU O.  3 5T x 85  If gl  wa C3  o  It .2 ^1 cl o a fl <u ©  0 0  m •>*  0 0  o  0 0  m co  0 0  o CO  0 0  m CM  0 0  o CM  0 0  m 1-  asuodsaj jopajap  o o o  o o m  o o in  CU fl u CU &U fl  54  V  B9JB 3S0||E /B3JB  9S010B|B6  ©  3 g g "3 "3  55  derivatization was done on fruit samples, reproducible and meaningful chromatograms could not be obtained. The allose peak area was about one quarter the size that from the standard curve chromatograms and it was a round peak with a flat top which was not similar to the peak produced when sugar mixtures of galactose and allose were derivatized (Figure 4). Moreover, there were multiple peaks eluted together around the galactose peak. A gas chromatogram of the same Spartan apple sample that had been spiked with 0.2 mg/ mL galactose showed that around the retention time of the galactose peak, there were two peaks with wide base connected together and again, the allose peak was not similar in shape and area compared to the chromatogram of mixture of galactose and allose (Figure 5). It was thought that there might be some other compounds that had co-eluted with galactose using a 30 m column; therefore a 60 m column was installed to try to provide more resolution and separation between the peaks. Although the peak resolution improved slightly, the peaks that eluted close to the galactose peak could not be separated and the galactose peak could not be clearly identified. Moisture in the dichloromethane phase was also thought to be causing interferences with the separations of peaks. Anhydrous sodium sulphate was added to absorb the moisture, but improvements to the chromatograms were not seen. Samples were passed through cation and anion resins in attempts to remove compounds that might interfere with the derivatization method but the resolution and peak shape were not improved. Studies have indicated that 500 pg of monosaccharides can be successfully derivatized by the amount of reagent used as described in Section 4.5.1. (Chaplin, 1994). However, up to 100 mg of monosaccharides in each sample (3 mL ethanol extract) was  56  <u tf  2  "a u '3 J3 * J  O  -M  fi T 3  CU _N  «  'u CU  Ti  vi  a «  c 8 c  1 0) E  u « a in B ©  U CQ  cu  X CU  W3 O  -a  cf  CJ  1s © £  s^ ©  fi  CQ S-  OS CJ  S-2  DC O  52  CQ S  e O  cu  ••*  -5 2 CU  ^ -is asuodsaj jojoaiap  So '2 • p-«  CU  57  T3  used in studies by Gross and Acosta (1991) and Gropper et al. (2000). This might be a possible reason for the strange shape and area of the allose and galactose peaks in the chromatograms produced by alditol acetate derivatization, due to incomplete derivatization of all the sugars present. However, experiments with a smaller amount of sample were not carried out to verify this assumption. 5.1.2. Trimethylsilylated-oxime (TMS-oxime) derivatives TMS-oxime derivatives were chosen as an alternative method due to the popularity of this method for determination of sugar concentrations in different fruits and the ease of procedures (Li and Andrews, 1986; Long and Chism III, 1987; Molnar-Perl and Morvai, 1992; Morvai and Molnar-Perl, 1992). It has been reported (Morvai and Molnar-Perl, 1992) that about 20 mg of carbohydrates could be successfully derivatized with the amount of derivatization reagents used as described in Section 4.5.2.. This equaled to the amount of sugars in 0.3 mL of ethanol extract from 10 g of samples. Therefore, TMS-oxime derivatization, which is a simpler and more reliable method, was selected to be used for all derivatizations in this research. A chromatogram showing 0.01 mg/ mL galactose, 1 mg/ mL of fructose, glucose and sucrose standards derivatized into TMS-oxime and TMS derivatives was shown in Figure 6. The concentrations of sugar solutions used correspond to 1 mg/ 100 grams fresh weight (gfw) of galactose and l g / 100 gfw of fructose, glucose and sucrose in fruits and vegetables used in this research (lmg/100 gfw of fresh fruit and vegetable was the lowest detection level used in this research). Moreover, an enlarged image of Figure 6 (Figure 7) indicates that the galactose peak (0.001 mg/ mL) is a sharp peak that can be quantified by peak integration, as seen in the standard curve (see below). Although 2  59  aj ce £ o . S3 !»% fc-  o •"t  es © ox> fl O CO. >-> fl  co  73 cy w> a  ^  '*  ce iT  e osx > flJ 2 S cu cu fl ce CM  ®  •s g  c u •—OX) fcS -  s 3 £ s  ce it  S  i-i o  C3 O  £ d fcw  co eg"  J  CU <u •fl ce CZ3  1  S H ^ 5  3  C3  "3 OX) <N  Iti H cu S  fl « S .2 g — w C 'Q 2 - S fc- — i i es S .. SO  -CU  °fl fl cu  asuodsgj jojoajap  fc- 0 _g fl '« _ ^  fl on!S T 3 fc-fl. *3 1 -2 CU  60  ta  OH  61  peaks were formed for each sugar, representing the syn and anti isomers, separation between the peaks were very clear and quantitation of galactose was achievable using the major peak. The minor peak for galactose was co-eluted with glucose's major peak whose retention time was very similar (Figure 8); whereas the two peaks for arabinose were eluted very close together (Figure 9). Using the major peak of galactose for quantification was also documented in research done by L i and Andrews (1986) and Molnar-Perl and Morvai (1992). L i and Andrews (1986) also documented the co-elution of galactose's minor peak with glucose major peak. A 10-point standard curve of concentrations from 0.001 mg/ m L to 0.06 mg/ mL galactose is presented in Figure 10. It shows that although the peak areas were small (Figure 6 and 7), they were still quantifiable and produced a linear response (response factor = 1.2307) with a high r value of 0.9906. Moreover, derivatization of sugars in 2  apple samples produced chromatograms that were repeatable and the peak area and the shape of the peak for the internal standard was constant with the peak area and shape produced from standard solutions (Figure 11). Although the galactose peak was small, an enlarged image (Figure 12) showed that the galactose from sugar extracted from fruit sample produced a sharp peak and the baseline around it was flat. The concentration of fruit sample or the amount injected into the G C column was not increased to amplify the galactose peak size. This is because fructose and glucose peaks, which surrounded the galactose peak, were very large. Increasing the concentration of the sample would also increase their peak areas, which might mask the galactose peak. Therefore, to keep the galactose peak visible, the concentration of the sample had to be adjusted to maximize the galactose peak, and at the same time, maintain  62  TS t. cQ TS  n  a> fi  CU  2  2  1  ^  xs cu 'K  in  & S  §".3 U  OJJ  CO.  >p  • s  3 C  -  E. v E  ft  a ^ J if S 3 I  s  .s © S i-i  TS cu cu u cn CQ -w  CQ  u ox)  ~53D  J if fi CQ hm  a fi o £  c?  £2 © gfl 2  E -5  i  CQ  o o LO  o o O •*  o o LO co  o o O co  o o LO CN  o o O CJ  o o L0  iasuodsaj jojooiap  o o o  o o LO  o o LO  2  <S  . fi  0CQ0 «« CU  2  .£f cu  ta  PH  63  CS cu  u  CS  cs  c M  cu o>  cu  ss g © s 2fi S 2  o © o co. c a  fe fl 53fi cu  in  -« "9, —1 r f  4*  WD CS  CU  S  E  ° fl  cu  a  T3 .fl A CU  S Vi  o © fl ce  "2 « CS « cs  M  cs  J2 n CS fl cu  « ? CM  '—5  ©2 S  E  I 8 © .s «-s s  o  u  A  fe cs  CS  CJ  l-H  CC  "  CS fl  O CU GSUOdSaj J0)33)3p  fe  cs  cu .2 CM  fl fl " ' OD cu  3  E 3 64  DC  ^-s © co ci  w  ^  ^ cs  'S ©  xi ^ XJ  M O  3  1 09 .83 CS CU  C  2  5  M  LO  ci  cu © B CA  — cy  o *-  X « ox) 2  ©3 exiO  o  •S  09  .2 £ CS  cs  M  ra E  _  >  OX)  •8 CU  O  O)  E,  |  ro ra  J3  OX) ©  .S  t>5  C §  a h  ci  © CJ  / - N  TJ  cu CJ  3  o  C3\ ON w  CM  ^ ^ CM  V©  O  .2 H i  CS  -O  09  X! O M  a CU > u  a 3 -s CS X ! CJ OX) 3 XI M CS X)  O CS  cu  s  cu CS CS 09 cu  o  eaje >|ead S I / B S J B >)ead |e6  CU  <Z3  a ©  • M CJ  •2 cu 73 .3  O  CU  M CS  t3  ~ CU  CJ  TS  C  fi cu2 « CM 5 3 CS OX) 3 2 M  .  ,  65  O  •  a -s  cu • .2 Qi 4-1  CS CQ. > -A  "S >-> «u fi "O CU  ft .  M  CU  ~ Tf  £  CU <N  fe  JS  lis  tZ3  DC « M  U o  r. O cs V  ^  te  CS  Hi ^  CS te  cs cu  73 ft  •  C O  • *>  ON u  cu *n ce f~~  .P  3 SO  T3  ar. C"U  cu w O ce  23  u  w  C  ^  ft fi ft  CS  CS  '—5 M 3 8 8 i, ~ cu W  S o ••  88  " s & .2 « -d cs cu cu cs  -M  -M  CU  ce  C  c cu  TS  M  CS CS v o ce CM  P  . ce  "3  3  5! -5  S3 g cs « M  8 to 'E — >•  P u s  I s "3 —  o M  JS U te CS  1  H -3  3  M  cu  -3 ce CU O  O £ § s* cu o >> ft ssuodsaj JOjoajap  M  So C/3  g  S ot H 3 66  asuodsaj jopajap  3 Cu  oc eg  ta  67  resolution between the peaks. Apple samples spiked with 0.0, 5.0, 10.0, 20.0 mg/100 gfw of galactose standard (Figure 13), added before extraction was carried out, were analyzed to determine the effectiveness of extraction, derivatization and quantification. From Figure 13, it could be observed visually on the chromatograms that as the concentration of added galactose increased, the galactose peak area increased accordingly. Table 1 revealed a 53.6% recovery of the galactose added to the spiked samples. It was thought that the major loss of soluble sugars was related to the extraction because the peak area of the internal standard, added after extraction and before derivatization, remained constant. The reduced effectiveness of the extraction step might be because samples were only washed once with 3 mL of 80% ethanol as stated by Gross and Acosta (1991) and Gropper et al. (2000), therefore large amount of sugars were left in the pulp rather than extracted. However, extractions for all samples were done before the recovery was determined, therefore methods could not be modified to improve sugar recovery. Furthermore, the % recoveries for different added galactose concentrations were not significantly different from each other (p= 0.05), therefore this derivatization method was suitable and acceptable for use in this research. 5.1.3. Problems in sampling method As described in Section 4.4., apple samples were randomly taken from 4-5 apples from each cultivar at each sampling time and the selected apples were peeled, cored, sliced and pooled together; subsequently three 10 g samples were taken from the mixed apple sample. Therefore, theoretically, there was only 1 sample with 3 replications taken. A trial was carried out with samples taken from individual apples to estimate the variance  68  s -2  S  ©X)  ON Cl, W  <»  • fa  CJ —  '5 S -2 s  fa  TT  «  a Si| 8  xi 8 Xi  I  fi  X3  CJ O  CQ 4>  2  cu cn M C3  J  - .I 1 & f aOX) - M» 5  5  ©S  o  ON O  QO  o  3^a  Z2.  ce  © CN  M A OX)  cu s M  So  CO «M  1  fa  0  S< B  ox).2 © 5M OX)  w  CU  I-  « ©  cS  cu  « 5 M xs o OXJXS  x> © fa. SS CJ  3  3 > S  I  OX) - M  38  5  1  ?  § « 1  w  Jjj CJ  CU  »  B  95  * ti S  •  M  W  —  M  - M  B  « S  a ^  i |  . "  ^  cu So  fa  CM  — lM Sr CU ^  I t 'S  0  fe g fa S .S  Table 1. The % recovery of different amounts of galactose standard added to stored Fuji apples (11 months at 4°C). Galactose (mg/lOOgfw) added  Galactose peak area  Internal standard peak area  5.77 5.77 5.77 10.09 10.09 10.09 21.84 21.84 21.84  1091 821 1044 1065 1178 1456 2205 2301 2468  9647 7260 9128 7365 8056 10085 9857 10130 10799 Averaged % recovery  Amount of free galactose detected (mg/lOOgfw) 8.67 8.66 8.76 11.07 11.20 11.06 17.14 17.40 17.51  % recovery  52.1 52.1 53.8 53.7 54.9 53.5 52.6 53.8 54.3 53.6 ± 1 . 3 1  1  Data is presented as mean ± standard deviation. The average amount of free galactose detected (before accounting for recovery) in Fuji apples stored for 11 months is 5.66 mg/ lOOgfw. % recovery was calculated by: (amount of free galactose in spiked sample (mg/lOOgfw) - 5.66mg/100gfw)*100 amount of galactose standard added (mg/lOOgfw) 1  averaged % recovery presented as average ± standard deviation  70  from samples that were not pooled (Table 2). Fuji apples that had been stored for 11 months were used, and three samples were taken from three individual apples. The analysis of the data for the 3 different apples showed that the standard deviation was only slightly higher than that reported for the pooled samples analyzed for the storage study (Section 5.2). Therefore, although the variation would be expected to be slightly higher if samples had not been pooled, the results from this research would have been similar. Pooling of samples posed a problem in statistical analysis, as no analysis could be done when n=l. Therefore, to factor in the individual apple differences and differences among samples, statistical analysis on results for the storage study was done with 3 readings: 1) mean of the pooled sample, 2) mean plus 2 times standard deviation from the individual apple study (0.90 for free galactose, 0.15 for free arabinose and 1.6 for sucrose) and 3) mean minus 2 times standard deviation from the individual apple study. 2 times the standard deviation was used to take account of the highest possible variation that can come about if 3 separate pooled samples were analyzed.  71  Table 2. Free galactose content of Fuji apples that had been stored for 11 months in regular atmosphere at 4°C.  Sample #  Galactose peak area  IS peak area  Amount of free galactose (mg/lOOgfw)  A A A B B B C C C  984 683 642 561 904 923 578 482 662  13305 8842 8353 7809 12998 13432 8555 7023 9441  11.27 11.23 11.17 10.73 10.60 10.47 10.10 10.26 10.19 Average (among samples)  Average free galactose content (mg/ lOOgfw) (within sample)  C.V. (%)  11.00±0.31  2.8  10.36±0.22  2.2  10.18±0.08  0.79  10.5L+0.43  4.1  1  2  Data is presented as mean ± standard deviation. Three samples (A, B , C) (n=3) consisting of 1 Fuji apple each were used. Each sample was analyzed in triplicate for their free galactose content. C.V. = coefficient of variation average free galactose content (within sample) was calculated with the 3 replications done for the same sample. average of free galactose content among samples was calculated with the average free galactose contents of the 3 samples. 1  2  72  5.2. Storage study of different varieties of apples Storing apples in a chilled environment after harvest is a common practice to delay ripening so that fruits can be marketed at later times. In this study, the effects of cultivar difference and storage on the apples' free galactose contents were examined by monitoring the change in amount of free galactose in 4 different apple cultivars during 9 months under cold storage at 4°C in regular atmosphere. 5.2.1. Conditions of apples during storage A l l apples softened as storage progressed. The poor storing apples (Spartan and Red Delicious) softened more quickly than the good storing apples (Braeburn and Fuji), however, after 6 months of storage, the softness of all apple cultivars were thought to affect the eating quality. Some apples also developed brown pits just under the skin as storage time increased. Rotting by fungi on some apples was also evident after 6 months of storage. Circular and light brown rots are often produced by infection with Gloeosporium album and G. perennans (Ministry of Agriculture, Fisheries and Food, 1979). Any apples that displayed any kind of drying, brown pits, brown core or rotting were removed from storage. 5.2.2. Free galactose in apples Before storage, Red Delicious apples (4.86 mg/ 100 gfw) had the lowest free galactose content followed by Fuji and Braeburn. Spartan had the highest free galactose content before storage (7.26 mg/100 gfw) which was shown to be significantly different from Red Delicious apples. As the apples were stored, each cultivar exhibited changes in free galactose contents during the storage time that were all significantly different from one another. This was confirmed when a significant interaction of cultivar and time was  73  Table 3. Free galactose content (mg/ 100 gfw) of 4 different apple cultivars stored for 0, 3, 6, 9 months in regular atmosphere at 4°C. Apple cultivar Braeburn (mg/ 100 gfw) C V . (%) Spartan (mg/100 gfw) C V . (%) Fuji (mg/ 100 gfw) C V . (%) Red Delicious (mg/ 100 gfw) C V . (%)  0 6.96 2.4 7.26 2.1 5.04 2.4 4.86 1.5  ±0.17  Storage time (months) 3 6 7.57 ±0.11 7.85 ±0.16  9 7.28 ± 0.29  ±0.15  2.0 10.80 ±0.11  1.4 10.99  4.0 12.40 ±0.14  ±0.12  0.9 5.63 ± 0.044  1.9 9.15  ± 0.072  0.79 6.90 ±0.18  1.4 6.83  2.6  2.9  awxy  awx  awxy  awy  awxy  aw  bx  aw  aw  bwx  bwxy  awy  ± 0.21  ±0.12  ±0.19  aw  bx  1.1 8.66  bwy  ± 0.026  0.30 4.67 ± 0.065 az  1.4  Three subsamples (n=3) were analyzed for each cultivar at each sampling period, and results were presented as mean ± standard deviation. Significant differences among cultivars and among sampling times were determined by one-way A N O V A and Tukey's multiple comparison test based on 3 readings consisting of the mean and mean ± 0.90, which is 2.1 times the standard deviation obtained from the individual apple study discussed in Section 5.1.3. For explanation of statistical analysis, refer to Section 5.1.3. and 4.6. C.V.= coefficient of variation " treatments denoted by different superscripts are significantly different from each other within the same cultivar (within each row) (p=0.05). " treatments denoted by different superscripts are significantly different from each other within the same storage time (within each column) (p=0.05). a  w  d  z  74  observed (p<0.000) (Table 4). Braeburn and Red Delicious apples did not show any significant change in free galactose concentrations throughout the storage period; whereas Spartan showed increase after 3 months of storage and remained at the same level after that. Fuji apples also showed increase in free galactose content after 6 months of storage and the amount persisted after 9 months of storage. (Table 3 and Figure 14). There are only a few reports (Gross and Acosta, 1991; Gropper et al., 2000) that have evaluated the amount of free galactose in fruits and vegetables. Gross and Acosta (1991) analyzed the soluble galactose content of 45 fruits and vegetables, including apples. They reported that apples have 8.3 mg/ 100 gfw of free galactose. However, they did not specify the variety and physiological age of the fruit tested. The amount of free galactose reported in this study is in the range of 4.86 mg (in Red Delicious at time 0) to 12.40mg/ lOOgfw (in Spartan after 9 months of storage), which encompassed the literature value. In this study, where the amount of free galactose in plant tissue was evaluated, it was thought that the quantities of other sugars could be used to validate the results. This is because of the difficulty in analyzing the small amounts of free galactose in fresh fruits and more importantly, it could be used to support the free galactose results observed in this study. The changes in free arabinose, which is also a monosaccharide that is attached to pectin side chains, and sucrose, the main free sugar in fruits, were monitored to determine if these sugars followed the same varietal trends and storage trends over time as in free galactose.  75  Table 4. 2-way analysis of variance of free galactose content in 4 apple cultivars stored in regular atmosphere at 4°C for 9 months. Samples from each cultivar were taken after 0, 3, 6, 9 months of storage.  1  Cultivar  Degrees of freedom 3  Time  Cultivar x time  Free galactose content F-ratio P-value  54.6 0.000  3  F-ratio P-value  19.7 0.000  9  F-ratio P-value  6.74 0.000  1  Values are considered to be significantly different at p< 0.05.  A N O V A was based on 3 readings consisting of the mean and mean ± 0 . 1 5 , which is 2.1 times the standard deviation obtained from the individual apple study discussed in Section 5.1.3. For explanation of statistical analysis, refer to Section 5.1.3. and 4.6.  76  5.2.3. Free arabinose in apples Arabinose is another major sugar that is attached to the side chains of pectins. Studies (Nara et al, 2001; Fischer et al, 1994; Massiot et al, 1996) have reported on the loss of arabinose from pectin side chains and other cell wall materials along with galactose during storage. Since the major source of arabinose is bound and free arabinose has little nutritional significance, there is limited information on the amount of free arabinose in plant tissues. In this study, free arabinose content in all cultivars increased continuously up to the end of 3 months of storage in 4°C and leveled off after 3 months of storage (Table 5; Figure 15). Spartan apples had the highest amount of free arabinose at the beginning of the storage trial and Red Delicious had the lowest. Fuji apples had the most amount of free arabinose at the end of 9 months of storage and Spartan had the least. Although all cultivars showed the same general pattern where free arabinose content increased up to 3 months and leveled off thereafter, they were shown to be significantly different from each other at all times by 2-way A N O V A (Table 6) as a significant difference was shown between the interaction of cultivar and time (p< 0.000), indicating that in fact, different cultivars showed different trends in changes in free arabinose content over storage time. It should also be noted that amounts of free arabinose were presented as peak area ratios since a standard curve for arabinose was not performed to determine arabinose's response factor with reference to the internal standard (phenyl-(3-D-glucopyranoside).  78  Table 5. Free arabinose content expressed as peak area ratio (peak area of arabinose/ peak area of IS) of 4 different varieties of apples stored at 4°C in regular atmosphere for 0, 3, 6, 9 months. Apple cultivar Braeburn (arabinose peak area/ IS peak area) C V . (%) Spartan (arabinose peak area/ IS peak area) C V . (%) Fuji (arabinose peak area/ IS peak area) C V . (%) Red Delicious (arabinose peak area/ IS peak area) C V . (%)  0 0.276 ± 0.0037  Storage time (months) 6 3 bwxy 1.46 ± 0.0097 0.038  1.4 0.331 ± 0.0048  1.1 0.655 0.031  1.4 0.302 ± 0.0021  4.7  0.70 0.253 ± 0.0040  0.75 0.815 0.027  1.6  3.4  aw  aw  aw  aw  Q  1  8 9 2  0  g  cwxy  ±  bwxy  b w x  ±  0.0081  bwy  ±  cwxyz  ±  2.6 1.39 ± 0.015  2.7 1.21 0.061  ±  1.1 1.65 0.022  5.1 1.60 0.041  ±  1.3 1.87 ± 0.030  2.6 1.62 0.047  ±  1.6  2.9  cwx  cwyz  ±  9 1.36 0.037  cyz  ±  cwxy  cwyz  cwyz  Three subsamples (n=3) were analyzed for each cultivar at each sampling period, and results were presented as mean ± standard deviation. Significant differences among cultivars and among sampling times were determined by one-way A N O V A and Tukey's multiple comparison test based on 3 readings consisting of the mean and mean ± 0.15, which is 2.1 times the standard deviation obtained from the individual apple study discussed in Section 5.1.3. For explanation of statistical analysis, refer to Section 5.1.3. and 4.6. C.V.= coefficient of variation treatments denoted by different superscripts are significantly different (p=0.05) from each other within the same cultivar (within each row). " treatments denoted by different superscripts are significantly different (p=0.05) from each other within the same storage time (within each column). a d  w  z  79  tu M  73 73 CU OH CU  ce  fi .© "-M  73  • p-t  > cu  -3 -3 73 -3 M  cu cu 1: J  D  S  ce +1  Hit  cu fi cu  73  cu E  ce ce S7 S3  2®  S -fi ce CS  cu  L. CU CS M  c o  E,  O E u  O)  ra k . o  to  CU  ce c O  C M  cu a, •B 3 fi  cs  U  ce  ©Q CS  cu -  1  cs Cm  ce .5  ° fe CS « M  ^  CS Tf  1-9 cS  M  - cu S fe — CS  cu _^ 2 cu  1  *  CM  ©  M  « "2 co co ci ci 6 3 J B ^ead s i /eaje >(ead asomqeje  CM  ci  d  ~3  W  e  ir  ce  . -2 (  S  1  3D 2  E .S80  Table 6. 2-way analysis of variance of free arabinose content (as peak area ratio) in 4 apple cultivars stored in regular atmosphere at 4°C for 9 months. Samples from each cultivar were taken after 0, 3, 6, 9 months of storage.  1  F-ratio P-value  Free arabinose content (peak area ratio) 18.7 0.000  3  F-ratio P-value  425 0.000  9  F-ratio P-value  5.69 0.000  Cultivar  Degrees of freedom 3  Time  Cultivar x time  1  Values are considered to be significantly different at p< 0.05.  A N O V A was based on 3 readings consisting of the mean and mean ± 0 . 1 5 , which is 2.1 times the standard deviation obtained from the individual apple study discussed in Section 5.1.3. For explanation of statistical analysis, refer to Section 5.1.3. and 4.6.  81  5.2.4. Sucrose in apples Sucrose, which is a major sugar exist in free form in fruits and vegetables, was also used to validate the changes in carbohydrate content as the fruit ripened during storage and to strengthen the results in free galactose content over time. The major role of sucrose in fruits and vegetables is as a storage compound and as an energy source (Hawker, 1985). In this study, sucrose contents were also presented as peak area ratios because the peak area of sucrose was beyond the galactose standard curve concentration range; moreover a sucrose standard curve with the appropriate internal standard had not been performed to determine the response factor for sucrose. When the peak area ratio of sucrose to internal standard was analyzed, a general continuous decreasing trend was seen for all cultivars (Table 7 and Figure 16) and the decreasing trend was shown to be cultivar specific when analyzed by 2-way A N O V A (p= 0.049)(Table 8). This decrease in sucrose in apples during storage was also reported by other researchers (Mahajan, 1994; Fuleki et al, 1994; Drake and Eisele, 1999). It was suggested that sucrose is used as a metabolite for respiration and other physiological processes, and is not replenished during storage. Hence, a decline in sucrose content was expected.  82  Table 7. Peak area ratio of sucrose (sucrose peak area/ internal standard peak area) as a TMS derivative of 4 different varieties of apples stored at 4°C in regular atmosphere. Apple cultivar Braeburn (sucrose peak area/IS peak area) C V . (%) Spartan (sucrose peak area/ IS peak area) C V . (%) Fuji (sucrose peak area/ IS peak area) C V . (%) Red Delicious (sucrose peak area/ IS peak area) C V . (%)  0 16.8 " ± 0.66  Storage time (months) 6 3 14.7 ±0.19 1 4 . 3 ± 0.080  9 10.4 ± 0.50  4.0 16.8 ±0.47  1.3 14.0 ± 0.30  0.55 9.78 ±0.10  4.8 5.47 ±0.18  2.8 10.5 ±0.25  2.2 9.33  ± 0.36  1.1 8.45  ±0.18  3.2 5.44 ± 0.070  2.4 11.3 ±0.38  3.8 9.01 ±0.35  2.2 5.59  ± 0.078  1.3 3.32 ± 0.069  3.4  3.8  1.4  ab  aw  ax  ax  abw  abcw  aw  abx  abx  bx  abxy  bcy  CW  cx  bx  cx  2.1  Three subsamples (n=3) were analyzed for each cultivar at each sampling period, and results were presented as mean ± standard deviation. Significant differences among cultivars and among sampling times were determined by one-way A N O V A and Tukey's multiple comparison test based on 3 readings consisting of the mean and mean ± 1.6, which is higher than double the standard deviation obtained from the individual apple study discussed in Section 5.1.3. For explanation of statistical analysis, refer to Section 5.1.3. and 4.6. C.V.= coefficient of variation treatments denoted by different superscripts are significantly different (p=0.05) from each other within the same cultivar (within each row). " treatments denoted by different superscripts are significantly different (p=0.05) from each other within the same storage time (within each column). a d  w  z  83  te fe a  84  Table 8. 2-way analysis of variance of free sucrose content (as peak area ratio) in 4 apple cultivars stored in regular atmosphere at 4°C for 9 months. Samples from each cultivar were taken after 0, 3, 6, 9 months of storage.  1  Cultivar  Degrees of freedom 3  Time  Cultivar x time  Free sucrose peak area ratio F-ratio P-value  43.7 0.000  3  F-ratio P-value  50.5 0.000  9  F-ratio P-value  2.20 0.049  1  Values are considered to be significantly different at p< 0.05.  A N O V A was based on 3 readings consisting of the mean and mean ± 0.15, which is 2.1 times the standard deviation obtained from the individual apple study discussed in Section 5.1.3. For explanation of statistical analysis, refer to Section 5.1.3. and 4.6.  85  5.2.5. Effect of varietal differences and storage on the amount of free galactose, arabinose and sucrose in apples It is well established that cultivar affects the amount of total sugars as well as the proportion of individual major sugars in apples (Fuleki et al, 1994; Fourie et al, 1991). The data presented here supported this view in terms of free galactose content and peak area ratios of arabinose and sucrose presented at the start of the storage trial. As well, all cultivars tested showed different trends in the content of free galactose as the fruits were stored. In addition, 2-way A N O V A results indicated that there is significant interaction between cultivar and time for all 3 sugars analyzed. Therefore, among the 4 cultivars tested, it is evident that varietal differences in apples do contribute to different amounts of free galactose in fresh and stored apples. Although soluble pectin or insoluble pectin content and composition was not analyzed in this research, other studies (Mahajan, 1994; Siddiqui et al, 1996; Fischer et al, 1994; Yoshioka et al, 1994; Redgwell et al, 1997) have indicated an increase in water-soluble pectin as the fruit is stored. Additionally, losses of galactose and arabinose from pectin side chains in apples during storage have been very well documented (Chun et al, 1999; Redgwell et al, 1997; Gheyas et al, 1998). In this study, it was found that free galactose of Spartan and Fuji ,and arabinose content of all cultivars increased during the earlier part of the storage and then the concentrations tapered off, whereas sucrose decreased throughout storage. Therefore, it is likely that galactose and arabinose were liberated from pectin side chains during ripening and storage and remained in free form after they were released from the pectin side chains. This explains the initial increase in free galactose and arabinose seen in all cultivars during storage. The release of galactose  86  and arabinose residues from pectin side chains is likely due to the action of cell wall hydrolases whose activities increase during fruit ripening and storage. However, as the storage time progresses, the activities of cell wall hydrolases are reported to decrease (Seymour and Gross, 1996; Fischer and Bennett, 1991; Fry, 1995). This might explain why during the latter part of storage, the level of free galactose and arabinose decreased. As galactose and arabinose are not released from pectin side chains anymore and like other sugars, continue to be utilized by fruit as fuel to maintain respiration and other functions, a net loss in free galactose and arabinose is seen. Another factor that may have contributed to the decrease in free galactose amounts during the latter part of storage is the action of cell wall synthesizing enzymes whose activities are highest during the latter part of storage (Fry, 1995; Seymour and Gross, 1996). Galactose liberated from pectin side chains by cell wall hydrolases are incorporated into other polysaccharide chains by these cell wall synthesizing enzymes; therefore the galactose may have become bound again. However, these cell wall synthesizing enzymes are not very well characterized and documented, therefore, further work is required to confirm this hypothesis. Braeburn and Red Delicious apples did not show any significant changes within the 9-months storage. This might be due to the low activities of cell wall hydrolases or the continuous action of cell wall synthesizing enzymes that could reshuffle the liberated galactose from pectin chains into other cell wall polysaccharides even during the early part of storage. However, no study has been done to compare the enzymatic composition and activity profiles of different apple cultivars during storage. It is generally believed that different cultivars possess different types of enzymes that results in different fruit ripening patterns and compositional differences.  87  5.3. Thermal treatments of apples and green beans The amounts of free galactose in Fuji apples and green beans after blanching and canning were evaluated in this study. Apple was chosen as an example of high acid product that receives pasteurization heat treatment while canned and green bean is an example of low acid product that is sterilized when canned. 5.3.1. Lethality calculation The amount of lethality each can received was calculated by the improved general method (IGM) and is shown in Table 9. The P value for canned apples was calculated using z= 16 F° and reference temperature of 212°F and the F for canned green beans 0  was calculated using z =18 F° and reference temperature of 250°F, which is the z value for Clostridium botulinum. Apples canned at 212°F for 20 minutes according to recommended thermal program had a P value of 10.68 minutes. Moreover, canning apples for 38 minutes at 212°F gave a P value of 21.59 minutes, which doubles the lethality obtained from the recommended thermal program. Canning green beans at 250°F for 11 minutes (recommended thermal program) gave a F value of 4.98 minutes 0  which is above the minimum of 3 minutes for low acid foods. Green beans canned for 16 minutes at 250°F gave a F = 10.29 minutes, which double the lethality obtained from 0  canning with the recommended process time. The thermal processes carried out, therefore, met the requirements of this study of having canned products that were canned to commercial sterility and double the commercial sterility for both low acid and high acid foods.  88  Table 9. Amount of lethal heat treatment canned apple slices and canned green bean received calculated with Improved General Method.  Apple slice (canned to commercial sterility) Apple slice (canned to double commercial sterility) Green beans (canned to commercial sterility) Green beans (canned to double commercial sterility)  Net weight (g)  Total lethality (minutes)  623.9 ± 6.27  P= 10.7 ± 0 . 5 1  20  625.1 ±5.81  2P= 21.6 ± 0 . 4 3  38  656.2 ±6.81  F = 4.98 ± 0 . 2 1  11  655.0 ±7.19  2F = 10.3 ± 0 . 1 3  16  1  Process time (minutes)  0  0  Values are presented as mean ± standard deviation. 2 samples from each canning session (n= 3) were used to calculate the lethality and to determine net weight in each product. Pasteurization values (P and 2P) for canned apple slices were calculated using z of 16 F°. z of 18 F° was used for calculation of F and 2F for canned green beans. 0  1  0  Total lethality (FJ P) was calculated using the Improved General Method. Total lethality= £ l 0 " * At T= temperature of the cold spot in can (°F) T = reference temperature (°F) At = change in time (minutes) ( T  T r ) / z  r  89  5.3.2. Free galactose, arabinose and sucrose concentrations in Fuji apples after thermal processing In this study, the free galactose content in blanched and canned apples was compared to fresh apples that had been stored for 9 months (Table 10). Blanching reduced the amount of free galactose in fresh Fuji apples from 8.66 mg/100 gfw to 2.78 mg/100 gfw. Canning the apples to commercial sterility further reduced the amounts of free galactose in the apples to an undetectable level (<1 mg/100 gfw). However, when the apples were canned to achieve heat treatment equaled to double the P value, 3.45 mg/ 100 gfw of free galactose was found, which was more than the amount found in blanched apple pieces. Free arabinose content in apples also showed the same trend as free galactose content (Table 10). The amount of free arabinose (expressed as peak area ratio) also declined after blanching (from 1.60 to 0.962) and was further decreased to 0.734 when apples were canned to commercial sterility; moreover, the amount of free arabinose increased to 0.803 as the cans were processed to achieve double lethality. On the other hand, sucrose (expressed as peak area ratio) decrease as the severity of the heat treatment increased, although there was no significant difference (p= 0.05) between canning to commercial sterility and canning to double the commercial sterility. 5.3.3. Free galactose, arabinose and sucrose concentrations in green beans after thermal processing The free galactose, arabinose and sucrose contents in fresh green beans and after treatments with different heat processes showed similar trends as apple slices after thermal processing (Table 11). In this study, free galactose concentration of fresh green bean was found to be 1.92mg /100 gfw. In previous study by Gross and Acosta (1991),  90  Table 10. Free galactose, arabinose contents and sucrose peak area ratio in fresh, blanched and canned Fuji apples.  Before heat treatment  Type of heat treatment Canning to Blanching commercial sterility  Canning to double commercial sterility 3.45 ±0.17  Undetectable Free galactose 8.66 ± 0.27 2.78 ± 0.082 (mg/ lOOgfw) N/A 4.8 3.0 C V . (%) 3.1 0.803 ± 0.036 Free arabinose 1.60 ± 0.064 0.962 ± 0.032 0.734° ± 0.035 (arabinose peak area: IS peak area) 3.3 4.8 4.6 C V . (%) 3.6 2.36 ± 0.083 Sucrose 5.44 ± 0.25 3.36 ±0.15 2.47 ± 0.092 (sucrose peak area: IS peak area) 3.5 3.7 4.6 4.6 C V . (%) Data is presented as mean ± standard deviation. 3 separate pooled samples (n=3) were used and results were analyzed by one-way A N O V A where 3 averaged readings from the 3 separate pooled samples were used. Significant differences among samples were determined by Tukey's multiple comparison tests. a  a  a  c  b  d  b  b  c  c  C.V.= coefficient of variation " treatments denoted by different superscripts are significantly different (p=0.05) from each other (within each row). Undetectable levels= less than lmg/lOOgfw. a  d  91  Table 11. Free galactose, arabinose contents and sucrose peak area ratio in fresh, blanched and canned green beans. Before heat treatment  Type of heat treatment Canning to Blanching commercial sterility  Canning to double commercial sterility 7.95 ±0.31  Undetectable Free galactose Undetectable 1.92 ± 0.068 (mg/ lOOgfw) N/A N/A 3.8 C.V. (%) 3.5 Free arabinose 0.129 ± 0.0049 0.0922 ± 0.0466 ± 0.018 0.0891 ± (arabinose peak 0.0032 0.0032 area: IS peak area) 3.4 3.7 3.6 C.V. (%) 3.7 4.15 ±0.14 Sucrose 5.82 ±0.22 5.26 ± 0.24 6.94 ± 0.30 (sucrose peak area: IS peak area) 3.4 4.4 4.6 C.V. (%) 3.9 Data is presented as mean ± standard deviation. 3 separate pooled samples (n=3) were used and results were analyzed by one-way A N O V A where 3 averaged readings from the 3 separate pooled samples were used. Significant differences among samples were determined by Tukey's multiple comparison test. b  a  a  a  b  b  d  c  c  d  C.V.= coefficient of variation " treatments denoted by different superscripts are significantly different (p=0.05) from each other (within each row). Values are expressed as mean ± 1 standard deviation. Undetectable level= less than lmg/lOOgfw. a  d  92  they showed that green beans (Phaseolus vulgaris L.) had 4.3mg/ 100 gfw of free galactose. The difference might be due to cultivar differences and possibility due to different stage of maturity of the green beans. Free galactose content decreased after blanching to an undetectable level and was also undetectable after canning at 250°F for 11 minutes. Yet, after receiving heat treatment equivalent to F = 10.3 minutes, the amount of free galactose in the green beans 0  was found to be 7.95 mg/ 100 gfw, a concentration that is much higher than fresh green beans without any heat treatment. Free arabinose content (expressed as peak area ratio) also decreased from 0.129 in fresh green beans to 0.0922 after blanching and to 0.0466 when green beans were canned to achieve commercial sterility. The amount of free arabinose increased when green beans were canned to receive twice the amount of lethality to 0.0891. Sucrose concentrations (expressed as peak area ratio) in green beans decreased as the severity of the heat treatment increased. 5.3.4. Effects of thermal processing on the free galactose, arabinose and sucrose content in apples and green beans This study showed that free galactose, arabinose and sucrose contents were lost during blanching and canning to commercial sterility for both low p H (apple) and high pH (green beans) products. Blanching and canning to sterility may have decreased free galactose and arabinose due to diffusion of these water-soluble sugars into the blanching water. This effect was evident when the processing water in canned produce was analyzed for sugar content (Appendix B). The chromatograms of canned water were only for qualitative purpose because the amount of water used for blanching and the amount of blanching water added into each can were not accurately measured. Leaching of sugars  93  into processing water was also reported by Stolle-Smits et al. (1995), Nyman et al. (1993), Nyman et al. (1994) and Massiot et al. (1992). Studies have demonstrated blanching caused minimal modification to the structure of pectic substances (Massiot et al, 1992; Plat et al, 1988; Stolle-Smits et al, 1995; Muller and Kunzek, 1998; Nyman et al, 1993). Furthermore, Stolle-Smits et al (1995) analyzed the pectin composition of blanched green beans and found that there was only a small decrease in neutral sugars with blanching and majority of galactose and arabinose were still attached to the pectin chains after blanching. Therefore, it is likely that blanching only removed the free galactose and arabinose that were already present in the produce. Canning to commercial sterility caused an additional decrease in all the sugars analyzed in Fuji apples, and in arabinose and sucrose in green beans. The amount of free galactose in green beans was undetectable in both blanched and canned to commercial sterility samples, therefore, it was not possible to determine whether the amount in green beans had decreased or not. Additional decrease in sucrose might be due to leaching into processing water, and at this elevated heating condition, sucrose might also be hydrolyzed into glucose and fructose. It is difficult to determine if the loss of free galactose and arabinose from plant tissues observed during canning to commercial sterility in this study were originally in the free form or whether they were hydrolyzed from pectin side chains. Many studies have examined the changes in cell wall polysaccharide composition and structure after canning. It has been well established that pectin is partially solubilized and lost into the processing water during canning (Voragen et al, 1995; Thakur et al, 1997), whereas cellulose and hemicellulose were not soluble to any large extent. Loss of galactose and  94  arabinose from the pectin side chains that accompanies pectin solubilization has been observed in both low pH products like tomatoes (Reinders and Their, 1999) and high p H products like carrots (Massiot et al, 1992; Plat et al, 1988) and green beans (Nyman et al, 1994). Therefore, canning should increase the amount of free galactose and arabinose in the plant tissue. However, from our results, free galactose and arabinose were lost from plant tissue. This might be because the free galactose and arabinose that were in the free form and those detached from pectin side chains were also leached into the processing water. The net result is that the recovered free galactose and arabinose in the plant tissues were lower than the blanched samples. When apples and green beans were subjected to a more severe heat treatment (double sterilization), the free galactose and arabinose contents increased whereas, sucrose continued to decrease. Therefore, it is evident that some changes took place in the plant polysaccharides that caused the additional release of galactose and arabinose. As noted previously, it is known that heat treatment can cause partial solubilization of pectin polymers, and causes the release of galactose and arabinose from pectin side chains. This effect might be augmented by a more severe heat treatment as pectins can be further solubilized and depolymerized. Moreover, hemicellulose is reported to be degraded at a more rigorous heat treatment (Massiot et al, 1992). Therefore, galactose and arabinose that were attached to the hemicellulose polymers might also be released. However, the free galactose and arabinose were retained in the plant tissue instead of in the processing water. Some galactose and arabinose liberated from hemicellulose might be trapped inside the plant tissue. Cellulose and hemicellulose are bonded together to form a network around the cell (Vincent, 1999) and the galactose and arabinose that are  95  released during hemicellulose degradation might remain inside the cellulosic matrix. This effect was not observed in products canned to commercial lethality due to the fact that hemicellulose is not hydrolyzed and pectin degradation, mainly in the middle lamella, occurs outside the cell wall, therefore, released galactose and arabinose are solubilized into the processing water. At high temperatures, pectins degradation is caused by hydrolytic cleavage of the glycosidic bonds in the pectin backbone at low p H (pH< 4) and by the action of [3eliminative degradative reaction at higher pH (pH> 5). In this study, both low and high pH products had increased free galactose content after double sterilization heat treatment, but they cannot be compared against each other unless the amount of galactose still attached to pectin side chains are known. This would involve analyzing the pectin composition. Moreover, physiological age of the produce was thought to be important in affecting the amount of free galactose in canned products (see Section 5.4.4.).  96  5.4. Enzymatic aids used in juice production Enzymatic aids are widely used in the production of clear apple juice. In this study, the effect of enzymatic preparations that serve as clarification aids or as yield increasing aids were examined for their effect on free galactose content in clear apple juice produced. 5.4.1. Juice characteristics The total soluble solids, expressed as °Brix, pH and the yield of the 4 different types of Red Delicious apple juice produced are presented in Table 12. The juice obtained from control juice had a Brix value of 11.7 and p H of 3.9. This is very close to the reported values of other studies (11.4 °Brix and p H of 3.7 from Schols et al., 1991; 12.0 °Brix and p H of 4.1 from Ibarz, 1997), which also used Delicious apples for juicing. Juice produced from the addition of Ultrazym 100 was characterized with a Brix value of 11.8 and p H of 4.0, which was not significantly different from the control juice in terms of °Brix, p H and yield. The obtained °Brix and p H from this juice was also in agreement with other studies that have been done (12.0°Brix and p H of 3.7 from Schols et al., 1991). Clear apple juice produced from the addition of Pectinex Ultra SPL had higher refraction (12.9 °Brix) and lower p H value (3.7). This is also in accordance with the results presented by Schols et al. (1991), who found that juice produced with Pectinex Ultra SPL had 13.0°Brix and pH of 3.5. The increased in refraction and decrease in pH when compared to control juice with no enzyme preparation added and juice with clarification enzymes added indicated that more sugars and other soluble solids like soluble pectins and other fragments of cell wall were present in the juice due to increased depolymerization of cell wall materials during liquefaction (see section 5.4.3.).  97  Table 12. Total soluble solids (°Brix), pH and yield of Red Delicious juice produced by the addition of different enzymatic preparation aids.  Parameter °Brix at 20°C C V . (%) pH at 20°C C V . (%) Release fluid yield 1  Enzymatic preparations added + Pectinex + Ultrazym 100 Ultra SPL  None  + Pectinex Ultra SPL and Ultrazym 100  11.7 ±0.42  12.9 ±0.31  11.8 ±0.49  13.1 ±0.44  3.5  2.4  4.2  3.3  3.9 ± 0.06  3.7 ±0.06  4.0 ± 0.06  3.7 ± 0.06  2  2  2  2  81.7 ± 3.1  155 ± 6.1  84.3 ± 4.0  161 ± 5.0  a  a  a  b  b  b  a  a  a  b  b  b  C V . (%) 3.8 4.0 4.8 3.1 Data is presented as mean ± standard deviation. 3 separate pooled samples (n=3) were used and results were analyzed by one-way A N O V A where 3 averaged readings from the 3 separate pooled samples were used. Significant differences among samples were determined by Tukey's multiple comparison tests. C.V.= coefficient of variation yield was expressed as amount of juice produced (mL) per 400g puree. " treatments denoted by different superscripts are significantly different (p=0.05) from each other (within each row). Values are expressed as mean ± 1 standard deviation. 1  a  b  98  Furthermore, liquefaction produced juice that was higher in acidity due to the release of free galacturonic acids from pectin chains (see section 5.4.3.). Addition of both enzyme preparations produced juice that had similar Brix and p H of juice where only Pectinex Ultra SPL was added. Juice yield was recorded as the amount of juice (mL) per 400 g of apple puree used. The design of this experiment where juice was not produced by pressing the pomace but only by dripping might underestimate the amount of control juice produced. The pulp that was left after expression of crude juice was wetter than the pulp with added Pectinex Ultra SPL. However, it is likely that the amount of juice produced from addition of Pectinex Ultra SPL still would be greater than from control juice even with pressing. The addition of Ultrazym 100 did not increase the amount of juice produced compared to juice with no enzyme preparation added. The similarity in juice yield between the two types of juices was expected as Ultrazym 100 was added after the crude juice had been expressed from apple puree. The addition of Pectinex Ultra SPL dramatically increased the amount of juice extracted from the same amount of apple puree due to the degradation of all cell wall materials, therefore reducing the ability of cell wall materials to form gels with water, thus "liquefying" the pomace, facilitating juice release (Zadernowski, 1999; Will et al, 2000; Schols et al, 1991). 5.4.2. Free sugars contents in clear, amber coloured apple juice produced with different enzymatic preparations added The amount of free galactose, arabinose and sucrose in 4 different Red Delicious apple juices produced with different enzymatic pressing and clarification aids was  99  determined (Table 13). The addition of Pectinex Ultra-SPL (liquefaction enzyme preparation) dramatically increased the free galactose content in apple juice from 3.98 mg/100 mL in control apple juice to 18.60 mg/ 100 mL, whereas, the addition of the clarification aid, Ultrazym 100, caused a much smaller increase to 4.86 mg/100 mL. Amounts of free arabinose (expressed as peak area ratio) in the 4 types of apple juice showed the same trend as free galactose. Addition of Pectinex Ultra-SPL caused free arabinose content in apple juice to increase from 1.50 tol.83, while the addition of Ultrazym 100 caused a less drastic increase to 1.69 in free arabinose when compared to it's effect on free galactose levels. With the apple juice produced with both Pectinex Ultra-SPL and Ultrazym 100 added, an additive effect of each enzyme preparation was observed for both free galactose and arabinose content. The net increase of adding Pectinex Ultra-SPL (14.62 mg/100 mL for free galactose) and the net increase of adding Ultrazym 100 (0.86 mg/100 mL for free galactose) are added together, the sum (15.58 mg/100 mL for free galactose) are similar to the net increase in free galactose (15.33 mg/ 100 mL) and arabinose in the apple juice where both enzyme preparations were added. The amount of sucrose (expressed as peak area ratio) in all 4 juices were shown to be the same at p=0.05. It was expected that there would be no change in sucrose concentration because sucrose in apples exists in free form and sucrose was not a substrate for any of the enzyme preparations added. Moreover, this also indicated that the experimental conditions like heating, and agitation during juice production did not cause any alterations to sucrose concentration.  100  Table 13. Free galactose, arabinose contents and sucrose peak area ratio in clear apple juice produced with the addition of different enzymatic preparations.  None  Enzymatic preparations added + Pectinex +Ultrazym 100 Ultra SPL  J  +Pectinex Ultra SPL and Ultrazym 100 19.3 ±0.20  Free galactose 3.98 ±0.12 18.6 ±0.33 4.86 ±0.17 (mg/lOOmL) C V . (%) 4.4 3.4 3.9 3.3 Free arabinose 1.50 ± 0.056 1.83 ± 0.076 1.69 ± 0.055 2.04 ± 0.072 (arabinose peak area: IS peak area) C V . (%) 3.8 4.2 3.3 3.5 Sucrose 4 9 . 8 ± 1.6 5 0 . 3 ± 1.9 5 0 . 3 ± 1.9 5 0 . 5 ± 1.4 (sucrose peak area: IS peak area) C V . (%) 3.7 3.1 3.7 2.7 Data is presented as mean ± standard deviation. 3 separate pooled samples (n=3) were used and results were analyzed by one-way A N O V A where 3 averaged readings from the 3 separate pooled samples were used. Significant differences among samples were determined by Tukey's multiple comparison tests. 1  a  b  c  d  a  b  c  d  a  a  a  a  C.V.= coefficient of variation " treatments denoted by different superscripts are significantly different (p=0.05) from each other (within each row). Free galactose amounts were expressed as mg/ lOOmL of the single strength juice produced with different enzymatic preparations added Pectinex Ultra-SPL is a liquefaction enzymatic preparation. Ultrazym 100 is a clarification enzymatic aid. a  d  1  2  3  101  5.4.3. Effect of addition of different enzyme preparations to the free sugars concentrations in apple juice produced The production of clarified apple juice is an industry almost totally dependent on the use of added enzymes (Lae, 1995). In this experiment, the addition of liquefaction enzymes and clarification enzymes were studied. It was found that the addition of Utrazym 100, the clarification enzyme preparation, caused a slight increase in free galactose concentration, whereas the addition of Pectinex Ultra-SPL caused a dramatic increase in free galactose content in the clear apple juice produced. Although, the specific enzymatic activities present in both enzymatic preparations are not available from the supplier, other studies that have used Ultrazym 100 as a clarification aid have reported that it contains activities of pectinesterases, polygalacturonases and pectinlyases (Ibarz, 1997; Urlaub, 1996). As discussed in Section 2.7., Ultrazym 100, a clarification aid, only breaks down dissolved pectin in the juice and therefore, galactose and arabinose that are attached to insoluble pectin and other cell wall polysaccharides are not affected. The results of this study supported this observation. The slight increase in free galactose content in the juice, therefore, was believed to be due to enzymatic depolymerization of the dissolved pectin chains, thereby releasing the galactose that was attached to the dissolved pectins only. The addition of Pectinex Ultra SPL caused a large increase in free galactose content in the juice because it contains enzymatic activities of pectinases that can hydrolyze both dissolved pectin and undissolved protopectin (Urlaub, 1996). In addition, hemicellulases and cellulases are present that can further solubilize and partially depolymerize pectin and other cell wall polysaccharides (Dongowski and Sembries,  102  2001). Polysaccharides, oligosacchrides and monosaccharides released from cell wall materials contribute to the increase in Brix values. This complete breakdown of cell wall polysaccharides is termed total liquefaction (Will et al, 2000). As a result, not only the galactose and arabinose that are attached to pectin polysaccharides are released, galactose and arabinose that are attached to other cell wall materials like hemicellulose would also be released, therefore increasing the free galactose and arabinose contents in the juice produced. The juice produced from the addition of Pectinex Ultra SPL was cloudy, and the addition of Ultrazym 100 cleared the juice. This effect was seen to be additive which indicated that liquefaction by Pectinex Ultra SPL did not cause complete depolymerization of all cell wall materials. Instead, the solubilized pectin fragments kept proteins and other particles in suspension, thus producing turbidity in the juice. The use of the two enzyme preparations gives some indication about the position and availability of arabinose and galactose in cell wall materials. Pectinex Ultra-SPL increased free arabinose concentration 8% more than when Ultrazym 100 was used but increased free galactose concentrations 285% more than when Ultrazym 100 used. This may indicate that most of the arabinose exists in the dissolved portion of the pectin, while more galactose was found attached to insoluble pectins and hemicelluloses. Again, this is only a hypothesis as cell wall polysaccharides were not analyzed in this study, and the exact galactose and arabinose compositions of Red Delicious apple cell wall polysaccharides were not known.  103  5.4.4. Potential effect of physiological age of apples on juicing and canning It is thought that the physiological age of the produce might have an impact on the free galactose content on juice produced with the addition of clarification aid and on produce that is blanched and canned. In the study of storage effect on free galactose content, it was found that storage increased the amount of free galactose in apples due to the action of cell wall degrading enzymes. Furthermore, it is known that stored crops have more soluble pectin with galactose and arabinose containing side chains attached when compared to newly harvested fruits where insoluble pectin are abundant. As there is more soluble pectin in stored crops, the addition of clarification aid in juice production, which only hydrolyzes soluble pectin, would lead to more galactose to be released during clarification process. However, it is thought that physiological age of the fruit would not influence the amount of free galactose in the juice produced with total liquefaction with/without clarification step. This is because in total liquefaction all cell wall materials are degraded, which means it does not matter whether galactose is attached to soluble or insoluble pectin, they are still released when the pectin is degraded. The effect of physiological age of the apple can also influence the free galactose concentrations in blanched and canned produce. More free galactose and arabinose are present in the stored produce's plant tissue which could be solubilized into the blanching water during blanching. Consequently, stored crop has less bound galactose attached to cell wall polysaccharides which could be liberated during sterilization processing where insoluble pectins are degraded. New crop, on the other hand, has less soluble pectin and free galactose in plant tissue, so less galactose will be solubilized into blanching water.  104  Therefore, more bound galactose from insoluble pectin side chains could be released new crop undergoes sterilization heat treatment.  5.5. Implications of this work for dietary management for galactosemic patients From this research, it is known that varietal differences in apples do contribute to difference in free galactose content when the fruit is fresh and also after storage. However, this cannot be generalized to other fruits and vegetables. For example, different varieties of onions have similar carbohydrate compositions (Ng et al., 1998) and interestingly, the outer layers of an onion are found to have less free galactose than the inner layers. Therefore, not only different cultivars of a same product need to be examined for free galactose content, but also different parts of the product may need to be evaluated separately. It is thought that the analysis of free galactose content in different parts of a produce might be applicable to produce that have multi-layers, such as cabbage, celery and lettuce. Cold storage in regular atmosphere was shown to result in an increase in free galactose contents in Spartan and Fuji apples during fruit ripening and softening. However, significant changes in galactose concentrations during storage were not seen in Braeburn and red Delicious apples and the galactose content of Braeburn, Fuji and Red Delicious apples were all below 10 mg/100 gfw during the 9-months storage. It has been suggested that foods containing < 10 mg/100 gfw can be consumed liberally in a galactosemic patient's diet. Therefore, it is safe to recommend patients to eat any of the 4 cultivars tested early in the season. However, the free galactose concentration of Spartan apples exceeded the 10 mg/ 100 gfw limit after 3 months of storage, however, it is well below 20 mg/100 gfw, so can still be consumed in moderation. This study only evaluated apples from one season at one orchard therefore variations among seasons and different orchards have not been accounted for. Moreover,  106  other factors like different agricultural practices and seasonal weather conditions are known to affect sugar composition, storage life and quality of apples, therefore their influence on free galactose concentration and pectin composition of apples should be assessed. Furthermore, free galactose concentrations might be different in fruits stored in controlled atmosphere since the activities of several cell wall degradation enzymes including (3-galactosidase are retarded. This is presumably due to their requirements of O2 for their activities (Lau and Yastremski, 1991; Siddiqui et al, 1996). Hence, retardation of changes from insoluble pectin to soluble pectin was shown in a number of apple cultivars during C A storage (Marlett, 2000; Kovacs et al, 1997a). This might lead to reduced release of galactose from pectin side chains during storage, thereby free galactose levels of apples stored in controlled atmosphere might be less than those stored in regular atmosphere for the same amount of time. Blanching was shown to reduce the amount of free galactose in apples and green beans due to leaching of soluble components into processing water. Therefore, blanching in boiling water might be a sensible routine cooking practice for galactosemic patients. However, only apples and green beans were studied and other produce might have different responses to blanching heat treatment and might result in release of galactose from cell wall polysaccharides. Blanching would also result in loss of water-soluble vitamins and minerals and patients may have to supplement their vitamins and minerals intake. If vegetables are heated for too long, softening of the produce by degradation of cell wall materials including pectin and hemicellulose, the main sources of galactose,  107  would lead to release of galactose from side chains as the polysaccharides are degraded. For this reason, cooking practices like making stews and soups, which require boiling vegetables for a long time may increase the free galactose content in the food. This might be problematic when the cooking liquid is consumed together with the cooked vegetables. Similarly, canned food that are consumed with the processing liquid like apple and baby food puree may have higher free galactose concentrations. It was shown that canning to commercial sterility further reduced free galactose contents in apples or green beans, and that these products can be safely consumed when processing water is drained off. However, canned products can sometimes be reprocessed to ensure commercial sterility, and this would increase the free galactose concentration as seen in produce that are double processed. It might be appropriate to advise patients to avoid generic brands and lower grade canned fruits and vegetables since they are more likely to be over-processed, hence the reduced quality of the canned product. On the other hand, low grade canned fruits and vegetables are more likely to use over-ripe produce where protopectin and hemicellulose contents are less than immature fruits, therefore more free galactose are removed by blanching. However, it is still thought that these products should be avoided to minimize the possibility of consuming over-processed product. Furthermore, the thermal processes for canned foods in the market might be more severe than commercial sterility to ensure the safety or to alter the texture of the product (e.g. canned tomato). In doing so, galactose from other cell wall polysaccharides could be liberated and contribute to the increase in free galactose concentration as seen in produce that were double sterilized. Therefore other canned fruits and vegetables should be analyzed for their free galactose content before  108  recommendations can be made. In addition, other cooking practices like microwave cooking, steaming, pressure cooking and freezing on the degradation of cell wall polymers need to be investigated. Enzyme preparations added to aid in clear apple juice production increased the free galactose content in the juices produced. Addition of clarification aid caused a small but significant increase in free galactose content, while liquefaction aid caused a large increase. However, there is no way of knowing what enzymes are added during production of commercial clear apple juices. Therefore, consumption of clear juices should be limited. Moreover, other juices like fresh squeezed juices and cloudy juices have not yet been analyzed. To recapitulate, this research has shown that varietal differences and cold storage of apples do cause differences in free galactose concentrations, but the main effect of galactose release from cell wall polysaccharides were seen during thermal treatment and juice production. It is thought that the main cause of increased free galactose in fruits and vegetables is due to the release of galactose from pectin and hemicellulose due to degradation of cell wall polymers by either enzymes in vivo, exogenous sources of cell wall degrading enzymes or heat treatment. From this research, patients should be advised to choose apples that are fresh and to eat them raw or blanched. Processed apple products and canned green beans should be avoided mainly due to the fact that production methods used are not labeled and/ or known easily by the public, and these processing practices might alter cell wall polymers and cause the amount of free galactose to be increased. Yet, only apple products and canned green beans were evaluated in this study and other produce might display different characteristics in  109  changes in free galactose contents, therefore more research needs to be done to evaluate different products and other processing techniques that can cause degradation to cell wall polysaccharides.  110  6. Conclusion This study has evaluated the influence of cultivar, storage, thermal processing and enzymatic aids used in juice production on the free galactose concentration in apples and green beans. Free galactose contents in Spartan and Fuji apples increased after 3 and 6 months of storage respectively, whereas Braeburn and Red Delicious apples did not have significant increase in free galactose concentration throughout the storage period. Spartan had the highest free galactose concentration, whereas Red Delicious had the lowest before storage. Moreover, during the 9-month storage period, the 4 cultivars analyzed showed different characteristics in changes in free galactose concentrations. The increase in free galactose concentrations during storage may be due to release of galactose from pectin side chains as the fruit ripened. Cell wall synthesizing enzymes and respiration needs were also thought to contribute to the change in free galactose contents in apples over time. Free galactose concentrations in both low p H (Fuji apples) and high p H (green beans) products were shown to be reduced by blanching and canning to commercial sterility presumably due to solubilization of galactose into the processing water. When both products were canned to receive double lethality, free galactose concentrations were elevated. It is believed that galactose from pectic polysaccharides and hemicelluloses were released and the released galactose from hemicellulose were trapped inside the cellulose matrix. The addition of enzymatic preparations in the production of clear apple juice leads to escalation in free galactose concentrations. Addition of a liquefaction enzymatic aid  111  (Pectinex Ultra SPL) elevated the free galactose concentrations by nearly 5 times when compared to cold pressed juice; addition of a clarification aid (Ultrazym 100), on the other hand, only caused a slight increase (1.2 times). Liquefaction enzymes caused total breakdown of cell wall polysaccharides releasing galactose from pectin polymers and hemicellulose to be released. Clarification enzymes only act on soluble pectins, therefore lesser amount of galactose is released and exist in free form in the juice. The information obtained from this project can be used to assist in making dietetic recommendations for galactosemic patients. From the results, it is obvious that processed apple products should be used with caution especially for those where processing liquids are mixed together with the product (e.g. apple puree). Blanching is a possible way of reducing the amount of free galactose in apples, but its effects on other produce should be reviewed before recommending this to patients as a preferred cooking practice. 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